WO1999016452A1 - THERAPEUTICALLY EFFECTIVE 1α, 25-DIHYDROXYVITAMIN D3 ANALOGS - Google Patents
THERAPEUTICALLY EFFECTIVE 1α, 25-DIHYDROXYVITAMIN D3 ANALOGS Download PDFInfo
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- WO1999016452A1 WO1999016452A1 PCT/US1998/019862 US9819862W WO9916452A1 WO 1999016452 A1 WO1999016452 A1 WO 1999016452A1 US 9819862 W US9819862 W US 9819862W WO 9916452 A1 WO9916452 A1 WO 9916452A1
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- C07C2603/08—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing three- or four-membered rings
Definitions
- the current invention concerns novel analogs of l ⁇ ,25- dihydroxyvitamin D 3 which are agonists for both the slow genomic responses and agonists of rapid nongenomic responses and analogs which act solely as agonists or antagonists for the rapid nongenomic cellular responses in a wide array of diseases in which l , 25-dihydroxyvitamin D 3 or its prodrugs are involved.
- the invention concerns analogs depicted by the general formulae I-V.
- the invention additionally concerns a method for treatment of diseases caused by deficiency or overproduction of the vitamin D 3 metabolites.
- the current invention concerns therapeutic properties of l ⁇ , 25-dihydrox vitamin D 3 analogs which are selective agonists or antagonists for the genomic and rapid nongenomic cellular responses affecting calcium and phosphorus absorption, resorption, mineralization, collagen maturation of bone and tubular reabsorption of ⁇ phosphorus.
- the analogs of the invention are useful for treatment of bone diseases such as rickets, osteomalacia, osteoporosis, osteopenia, osteosclerosis, renal osteodystrophy; skin diseases, such as psoriasis; thyroid diseases, such as medullary carcinoma; brain diseases, such as Alzheimer's disease; parathyroid diseases, such as hyperparathyroidism, hypoparathyroidism, pseudoparathyroidism or secondary parathyroidism; liver and pancreas diseases, such as diabetes, cirrhosis, obstructive jaundice or drug-induced metabolism; intestine diseases, such as glucocorticoid antagonism, idiopathic hypercalcemia, alabsorption syndrome, steatorrhea, or tropical sprue; kidney disease, such as chronical renal disease, hypophosphatemic vitamin D-resistant rickets or vitamin D-dependent rickets; lung diseases, such as sarcoidosis; and for treatment of any other disease in which
- Analogs of vitamin D 3 metabolites act rapidly, specifically, and in the same manner as the vitamin D 3 metabolites on the genomic cellular apparatus and also elicit rapid nongenomic responses correcting the vitamin D 3 caused deficiencies.
- the certain analogs of l ⁇ ,25(OH) 2 D 3 have biological activities similar to those of l ⁇ ,25(OH) 2 D 3 without having undesirable secondary symptoms. Their biological activities are dependent on their respective chemical structures and these analogs are, therefore, more specific in their biological action. Some of these analogs act both as agonists of slow genomic responses and agonists of rapid responses while the others act solely as agonists or antagonists for rapid nongenomic responses.
- One aspect of the current invention is a compound depicted by the general formula I or a pharmaceutically acceptable salt thereof.
- Another aspect of the current invention is a compound of the formula I comprising substituents listed in Table 1.
- Another aspect of the current invention is a compound depicted by the general formula II or a pharmaceutically acceptable salt thereof.
- Another aspect of the current invention is a compound of the formula II comprising substituents listed in Table 2. Still another aspect of the current invention is a compound depicted by the general formula III or a pharmaceutically acceptable salt thereof.
- Another aspect of the current invention is a compound of the formula III comprising substituents listed in Table 3. Still yet another aspect of the current invention is a compound depicted by the general formula IV or a pharmaceutically acceptable salt thereof.
- Another aspect of the current invention is a compound of the formula IV comprising substituents listed in Table 4.
- Yet another aspect of the current invention is a compound depicted by the general formula V or a pharmaceutically acceptable salt thereof. Still another aspect of the current invention is the compound of the formula V comprising substituents listed in Table 5.
- Another aspect of the current invention is an analog selected from the group consisting of analog DE, DF, EV, HQ, HR, LO, JM (their names to be listed) , namely l ⁇ , 25 (OH) 2 -7-dehydrocholesterol; analog JN, namely, l ⁇ , 25 (OH) 2 -lumisterol 3 ; analog JO, namely, l ⁇ ,25(OH) 2 - pyrocalciferol 3 ; analog JP, namely, l ⁇ ,25(OH) 2 - isopyrocalciferol 3 ; analog HS, namely, l ⁇ , 18,25 (OH) 3 -D 3 ; analog GE, namely, 14-epi-l, 25 (OH) 2 -D 3 ; analog ⁇ GF, namely, 14-epi-l,25(OH) 2 -pre-D 3 ; analog JR, namely, 1 ⁇ , 25 (OH) 2 -7, 8- cis-D 3
- Still another aspect of the current invention is a process for preparation of analogs of general formulae I-V and salts thereof.
- Another aspect of the current invention is a method for treatment of diseases connected with or caused by vitamin D 3 deficiency or overproduction, by providing a subject in need of such treatment a vitamin D 3 analog which is either an agonist of vitamin D 3 or its antagonist, wherein the analog is selected from the group of compounds listed in Table 1.
- Still yet another aspect of the current invention is a method for eliciting slow genomic responses by interaction of the analogs of the invention with the nuclear receptor for l ⁇ ,25(OH) 2 D 3 which is present in target organ cells.
- Still yet another aspect of the current invention is a method for eliciting rapid nongenomic responses which include a rapid stimulation and release of calcium ions by intestine, kidney, parathyroid cells, liver and other organs during homeostatic responses and correction of pathological conditions in which the vitamin D 3 or l ⁇ ,25(OH) 2 D 3 are involved by analogs of the invention.
- Another aspect of this current invention is the rapid nongenomic stimulation of mitogen-activated protein kinase (MAP-kinase) in chick intestinal and human leukemic cells.
- MAP-kinase mitogen-activated protein kinase
- Still yet another aspect of the current invention is a method for rapid nongenomic stimulation of mitogen- activated protein kinase (MAP-kinase) in intestinal or leukemic cells by analogs of the invention.
- Still another aspect of the invention is a method for treatment of diseases caused by deficiencies or overproduction of l ⁇ ,25(OH) 2 D 3 or treatment of its functional deficiencies by providing a subject in need of correcting these deficiencies with an agonist analog of the l ⁇ ,25(OH) 2 D 3 represented by formulae I-V in amount sufficient to ameliorate the disease.
- Still another aspect of the current invention is a method for selective inhibition of vitamin D-related rapid nongenomic responses.
- Another aspect of the present invention involves controlling the rapid nongenomic responses mediated by l ⁇ ,25(OH) 2 D 3 by treating the subject in need of such treatment with an antagonist analog which is l ⁇ , 25 (OH) 2 D 3 .
- Another aspect of the current invention is l ⁇ , 25- dihydroxyvitamin D3 and its 6-s-cis analogs which are selective agonists for the activation of MAP-kinase.
- Another aspect of the current invention is a method for treatment of bone diseases such as rickets, osteomalacia, osteoporosis, osteopenia, osteosclerosis or renal osteodystrophy; skin diseases, such as psoriasis; thyroid diseases, such as medullary carcinoma; brain diseases, such as Alzheimer's; parathyroid diseases, such as hyperparathyroidism, hypoparathyroidism , pseudoparathyroidism or secondary parathyroidism; liver and pancreas diseases, such as diabetes, cirrhosis, obstructive jaundice or drug-induced metabolism; intestine diseases, such as glucocorticoid antagonism, idiopathic hypercalcemia, malabsorption syndrome, steatorrhea or tropical sprue; kidney disease, such as chronical renal disease, hypophosphatemic vitamin D-resistant rickets or vitamin D-dependent rickets; lung diseases, such as sarcoidosis; or any other disease ⁇ in which l
- Another aspect of the current invention is a method for treatment of vitamin D 3 deficiencies by providing l ⁇ ,25-dihydroxyvitamin D 3 analogs which are selective agonists or antagonists for the genomic and rapid nongenomic cellular responses affecting calcium and phosphorus absorption, resorption, mineralization, collagen maturation of bone and tubular reabsorption of phosphorus.
- Still another aspect of the current invention is a pharmaceutical composition
- a pharmaceutical composition comprising a l ⁇ , 25-dihydroxyvitamin D 3 analog useful for treatment of rickets, osteomalacia, osteoporosis, osteopenia, osteosclerosis, renal osteodystrophy, psoriasis, medullary carcinoma, Alzheimer's, hyperparathyroidism, hypoparathyroidism, pseudoparathyroidism, secondary parathyroidism, diabetes, cirrhosis, obstructive jaundice or drug-induced metabolism, glucocorticoid antagonism, idiopathic hypercalcemia, malabsorption syndrome, steatorrhea, tropical sprue, chronical renal disease, hypophosphatemic vitamin D receptor (VDRR) , vitamin D-dependent rickets, sarcoidosis.
- VDRR hypophosphatemic vitamin D receptor
- analog JM namely l ⁇ , 25 (OH) 2 7-dehydrocholesterol
- analog JN namely, l , 25 (OH) 2 -lumisterol 3
- analog JO namely, l ⁇ , 25 (OH) 2 -pyrocalciferol 3
- analog JP namely, l ⁇ ,25 (OH) 2 -isopyrocalciferol 3
- analog HS namely, l ⁇ ,18,25(OH) 3 -D 3
- analog GE namely, 14-epi-l, 25 (OH) 2 -D 3
- analog GF namely, 14-epi-l, 25 (OH) 2 -pre-D 3
- analog JR namely, l ⁇ ,25 (OH) 2 -7, 8-cis-D 3
- analog JS namely, l ⁇ ,25 (OH)
- Still yet another aspect of the current invention is a method for treatment of diseases which require rapid nongenomic stimulation and release of calcium ions by intestine, kidney, parathyroid cells, liver and other organs during homeostatic responses and correction of pathological conditions in which the vitamin D 3 or l ⁇ ,25(OH) 2 D 3 are involved by providing a subject in need thereof an analog of the invention.
- Still yet another aspect of the current invention is a pharmaceutical composition comprising an analog of the invention or its pharmaceutically acceptable salt.
- Step-like conformation the seco-B ring can assume, in the limit, one of two conformations as a consequence of rotation about the carbon 6-7 single bond; in the 6-s-cis orientation (C) the A ring is related to the C/D rings as in the conventional steroid orientation, referred to here as the "steroid-like conformation” and when the conformation is in the 6-s-trans orientation (D) , the A ring is present in an "extended conformation.”
- Alpha or “ ⁇ ”, “beta” or “ ⁇ ” position or configuration mean the absolute configuration notation used in steroids, such as cholesterol or in natural products; the term “ ⁇ ” or “ ⁇ ” mean the carbon or the substituent, as the case may be, within the context of the structural formulas presented herein.
- 6-trans orientation means a geometrical orientation resulting in an isomer having a spatial arrangement where a given atom positioned on each side of the carbon-carbon axis is in opposite location relative to the carbon axis.
- Ant means a compound capable of combining with receptors to initiate the compound's actions. The agonist possesses affinity and intrinsic activity.
- Antagonist means a compound which prevents, blocks, neutralize or impede the action of agonist.
- Conformationally flexible means analogs wherein a connection between a specified two carbons permits rotation of 360 degrees with respect to each other. Typically, two carbons exist in this configuration.
- Conformationally restricted means analogs wherein a connection between a specified two carbon does not permit rotation of 360 degrees with respect to each other. There is a degree of variability in conformationally restricted carbons. Two carbon in this context can, therefore, be more or less conformationally restricted and be able to rotate more or less.
- 6-cis-orientation means a geometrical orientation resulting in a spatial arrangement where a given atom, positioned on each side of the carbon-carbon axis is in the same side location relative to the carbon axis.
- 6-s-cis means, in this context, that there is a double bond between carbons C5-C6 and that C5-C6 carbons exist in fixed cis relation to each other.
- 6-trans-orientation means a geometrical orientation resulting in an isomer having a spatial arrangement where a given atom positioned on each side of the carbon-carbon axis is in opposite location relative to the carbon axis.
- Ant means a compound capable of combining with receptors to initiate the compound ' s actions . The agonist possesses affinity for the receptor.
- Antagonist means a compound that prevents, blocks, neutralizes or impedes the action of an agonist.
- l ⁇ ,25 (OH) 2 D 3 means l ⁇ , 25-dihydroxyvitamin D 3 .
- D 3 means vitamin D 3 .
- the official IUPAC name for vitamin D 3 is 9 , 10-secocholesta-5 , 7 , 10 (19) -trien-3 ⁇ -ol.
- VDR Transcaltachia
- VDR is a generic term that means l ⁇ ,25(OH) 2 D 3 receptors that include VDR nuc and VDR mem .
- VDR nuc means nuclear receptor for l ⁇ ,25(OH) 2 D 3 interacting with l ⁇ ,25(OH) 2 D 3 or with the analogs of the invention.
- VDR- em means membrane receptor for l ⁇ ,25(OH) 2 D 3 interacting with l ⁇ ,25(OH) 2 D 3 or with the analogs of the invention.
- Ligand means any small organic molecule that has a specific affinity for its cognate receptor.
- the ligand for the estrogen nuclear receptor is estradiol or its analogs.
- the ligand for the l ⁇ ,25(OH) 2 D 3 receptor, either VDR nuc or VDR- era is l ⁇ ,25(OH) 2 D 3 or its analogs.
- PMSF means phenylmethylsulfonyl fluoride.
- EGTA means ethylene-bis (oxyethylenenitrilo) - tetraacetic acid.
- HEPES 4- (2-hydroxyethyl) -1-piperazineethane- sulfonic acid.
- PKC protein kinase C
- MAP-kinase means itogen activated protein kinase.
- “Secosteroids” means compounds in which one of the cyclopentanoperhydrophenanthrene rings of the steroid ring structure is broken. In the case of vitamin D 3 , the 9-10 carbon-carbon bond of the B ring is broken generating a seco-B steroid.
- Rapid response or “rapid nongenomic response” means a rapid non-genomic effect of l ⁇ ,25(OH) 2 D 3 or analog thereof generated by interaction of l ⁇ ,25(OH) 2 D 3 or analog thereof with the membrane receptor, that is observed within seconds to minutes following the exposure of cells to these compounds .
- Gene response or “slow genomic response” means a biological response generated by interaction of ⁇ lo , 25 (OH) 2 D 3 or the analog thereof with the cell nuclear receptor resulting in the regulation of gene transcription. Slow genomic responses are observed within several minutes to several days.
- DBP means vitamin D binding protein.
- HRE hormone response element.
- Hormone response elements are composed of a specific sequence of about 6-12 nucleotides in the promoter region of the specific DNA constituting a gene which is regulated by steroid hormone receptors, including the nuclear receptor for l ⁇ ,25(OH) 2 D 3 .
- Target cell means any cell in the body that possess either membrane receptors (VOR-, ⁇ ) or nuclear receptors (VDR nuc ) for l ⁇ ,25(OH) 2 D 3 .
- Figure 1 illustrates a simplified version of the vitamin D endocrine system including the endocrine gland, the kidney which produces the two vitamin D related steroid hormones, and the categories of target organs where biological responses are generated and where vitamin D analogs function.
- Figure 2 illustrates both the central role of receptors for l ⁇ ,25(OH) 2 D 3 in mediating selective biological and the sites of action of both conformationally flexible and conformationally restricted analogs.
- Figure 3 illustrates the conformational flexibility of vitamin D molecules using l ⁇ ,25(OH) 2 D 3 as an example.
- Top view Figure 3A
- plane view Figure 3B
- rotational freedom Figure 3C
- Figure 4 illustrates the role of the vitamin D-binding protein (DBP) in mediating the delivery of l ⁇ ,25(OH) 2 D 3 or analogs to target cells.
- DBP vitamin D-binding protein
- Figure 5 represents a general model describing how l ⁇ ,25(OH) 2 D 3 and analogs of the invention, both conformationally flexible (Figure 5A and 5B) and conformationally restricted ( Figure 5C) , generate biological responses.
- Figure 5A and 5B conformationally flexible
- Figure 5C conformationally restricted
- Figure 6 illustrates mediation of the slow nuclear and rapid biological responses by l ⁇ ,25(OH) 2 D 3 and its conformationally flexible and conformationally restricted analogs with a correlation to potential target cells and therapeutical treatment modalities.
- Figure 7 presents results of the binding of l ⁇ ,25(OH) 2 D 3 and selected analogs to the vitamin D-binding protein.
- Figure 8 presents results of the binding of l ⁇ ,25(OH) 2 D 3 and selected analogs to the nuclear receptor for l ⁇ ,25(OH) 2 D 3 [VDR nuc ] .
- Figure 9 presents results of a classical in vivo biological assay in vitamin D-deficient chicks which quantitates the relative abilities of l ⁇ ,25(OH) 2 D 3 and selected analogs to stimulate an intestinal Ca 2+ absorption
- ICA bone Ca 2+ mobilizing activity
- BCM bone Ca 2+ mobilizing activity
- Figure 10 presents results from a cell culture assay which quantitates the relative abilities of l ⁇ ,25(OH) 2 D 3 and the analog HS to stimulate cell differentiation.
- Figure 11 presents results from a bioassay of transcaltachia, the rapid hormonal stimulation of intestinal Ca 2+ absorption, as stimulated by l ⁇ ,25(OH) 2 D 3 and selected analogs.
- Figure 12 presents typical results from a cell culture assay which quantitates the relative abilities of l ⁇ ,25(OH) 2 D 3 and selected analogs to stimulate mitogen-activated protein kinases (MAP-kinase) .
- MAP-kinase mitogen-activated protein kinases
- Figure 13 presents results from the assay of transcaltachia of the analog HL, namely l ⁇ , 25 (OH) 2 D 3 , to inhibit the rapid response of stimulation of transcaltachia by l ⁇ ,25(OH) 2 D 3 .
- Figure 14 illustrates the antagonist action of rapid responses elicited by treatment with l ⁇ ,25(OH) 2 D 3 and by analog HL.
- the present invention provides analogs of l ⁇ ,25(OH) 2 D 3 which are able to treat and ameliorate diseases and conditions connected with the vitamin D metabolism. These analogs effectively control gene expression via slow genomic responses as well as rapid nongenomic cellular responses typically mediated by l ⁇ , 2 ⁇ dihydroxyvitamin D 3
- the current invention therefore, relates to novel biologically active analogs of l ⁇ ,25 (OH) 2 D 3 .
- These analogs are agonists of slow genomic responses or selective agonists or antagonists of rapid nongenomic cellular responses, depending on their chemical structures.
- the group I is represented by compounds having a general formula I
- R x is CH 3 and when C_ and C3 are ⁇ - ⁇ , then R 2 is not the substituent I-l, 1-2, 1-3, 1-9 or 1-10; or when C ⁇ is in the ⁇ orientation and C3 is in the ⁇ orientation, C5-C6 double bond is cis or trans and C7-C8 double bond is trans, R x is CH 3 , C14 hydrogen is in the ⁇ orientation, C16-C17 is a single or double bond, then R 2 is not the substituent I-l, 1-2, 1-3, 1-4, 1-5, 1-9 or 1-10; or when C ⁇ is in the ⁇ orientation, C3 is in the ⁇ orientation, C5-C6 double bond is cis, C7-C8 double bond is trans, x is CH 3 , C14 hydrogen is in ⁇ orientation, C16-C17 is a single bond, then R 2 is not the substituent I-l; or when C x is in the ⁇ orientation, C3 is in the ⁇ orientation, C5
- the substituents I-l - 1-10 are the same as substituents II-l - 11-10, III-l - 111-10, IV-1 - IV-10 and V-l - V-10.
- the designation I, II, III, IV and V show the group of the compounds having the general formula I, II, III, IV or V to which the substituent selected form the substituents 1-10 is attached as R lf R 2 or R 3 .
- T ⁇ F Ri & R 2 refer to the substituents of general formula (I) or their suitably protected forms (R'j & R' 2 ) , usually as their silyl ethers; all structures may have single or double bonds across the C16-C17 positions.
- Reaction Scheme 1 illustrates preparation of compounds of the Group I. Compounds of the general formula I are chemically synthesized according to Scheme 1 using the three general approaches shown in Scheme 1-A, Scheme 1-B and Scheme 1-C.
- step 1 the vitamin D A-ring fragment 1 is treated in step 1 with butyllithium and then the resulting lithium salt is added to ketone 2 in step 2.
- step 2 The product from step 2 is reacted in step 3 with butyllithium and then benzoyl chloride to afford the propargyl benzoate 3.
- step 3 Reduction of 3 with samarium iodide with appropriate additives as in step 4 followed by deprotection with tetrabutylammonium fluoride (TBAF) in step 5 affords the 6 ⁇ -vinylallene analog 4.
- Photochemical irradiation as in step 6 affords the corresponding 6 ⁇ -vinylallene analog 5.
- Chromium (0) mediated isomerization of 4 in step 7 leads stereoselectively to the C5-C6 cis,C7-C8 cis analog 6, which upon photochemical irradiation using a medium pressure mercury lamp with triplet sensitizer in step 10 affords the C5-C6 cis, C7-C8 trans derivative 8.
- the vitamin D compounds 6 and 8 are converted by the same two step procedure (steps 8-9 or steps 11-12, respectively), to the corresponding C5-C6 trans compounds 7 and 9, respectively. Additional details for a specific case of the pathway of Scheme 1-A can be found in J. Am. Chem. Snr. 116, 6207-6216, (1994) .
- the analogs listed in Group I are represented by the analogs identified as HL, HH, HJ, HS, GE, DE, DF, HQ, HR, EV, JR, JS, JY, JX, LO and IB.
- the synthesis of these analogs is described in the Example 1 (DE) , Example 2 (DF) , Example 3 (EV) , Example 4 (GE) , Example 6 (HH) , Example 7 (HJ) Example 8 (HL) , Example 9 (HQ) , Example 10 (HR) , Example 11 (HS) , Example 12 (IB) , Example 17 (JR) , Example 18 (JS) , Example 19 (JV) , Example 20 (J ) , Example 21 (JX) , and Example 22 (JY) .
- These analogs depending on their structure, have a biological activity as agonists or antagonists of slow genomic responses and the rapid nongenomic responses.
- the antagonists of the Group I are represented by the generic formula I wherein R x is methyl, Cl hydroxyl is in ⁇ configuration, C3 hydroxyl is in ⁇ configuration, C14 hydrogen is in ⁇ configuration and R 2 is the substituent 2, 9, 10.
- the representative analog is the analog HL.
- the agonists of Group I are represented by a general formula I wherein R x is CH 2 OH, Cl hydroxyl is in ⁇ configuration, C3 hydroxyl is in ⁇ configuration, C14 hydrogen is in ⁇ configuration and R 2 are the substituents I-l - 1-10, preferably substituents 1-2, 1-3, 1-4, 1-9 and 1-10.
- the antagonist are compounds wherein R x is CH 2 0H, Cl hydroxyl is in ⁇ configuration, C3 hydroxyl is in ⁇ configuration, C14 hydrogen is in ⁇ configuration and R 2 are the substituents I-l - 1-10, preferably substituents 1-2,
- the group of agonists is represented by a general formula I wherein R 2 is CH 3 or CH 2 OH, Cl hydroxyl- is in ⁇ configuration, C3 hydroxyl is in ⁇ configuration, C14 hydrogen is in ⁇ configuration and R 2 are the substituents I-l - 1-10, preferably substituents I-l, 1-2, 1-3, 1-4, 1-9 and 1-10.
- the group of antagonists is represented by a general formula I wherein R x is CH 3 or CH 2 OH, Cl hydroxyl is in ⁇ configuration, C3 hydroxyl is in ⁇ configuration, C14 hydrogen is in ⁇ configuration and R 2 are the substituents I-l - 1-10, preferably substituents I-l, 1-2, 1-3, 1-4, 1-9 and 1-10.
- the group II is represented by compounds having a general formula II
- R x is not the substituent II-l.
- R ⁇ refers to the substituents of generic formula (II) or the suitably protected forms (R' _) ,usually as the silyl ether; all structures may have single or double bonds across the C16- 17 positions.
- Reaction Scheme 2 illustrates preparation of compounds of the Group II .
- each compound may have a single or double bond across C16-C17.
- the four general structures of compound 5 shown in Scheme 2 may be collectively represented by generic structure II.
- Scheme 2-A starts with the palladiu (0) mediated coupling of 1 with 2 in step 1 to afford 3a.
- Deprotection of the latter in step 2 using TBAF and THF gives 3b, which is followed by Lindlar semi-hydrogenation (step 3) affords the previtamin type compound 4.
- Heating previtamin 4 in step 5 at elevated temperatures as indicated affords the ⁇ and ⁇ isomers known as the pyrocalciferol and isopyrocalciferol types of vitamin D provitamins 5.
- step 4 photochemical irradiation through pyrex using a medium pressure mercury lamp affords the 9 ⁇ ,10 ⁇ , and the 9 ⁇ ,10 ⁇ provitamin D type isomers known as the dehydrocholesterol and the lumisterol analogs 5.
- the A-ring phosphine oxide 6, after deprotonation in step 6, is coupled with CD ring fragment 7 in step 7, a so-called Horner-Wittig reaction.
- the resulting product is 8 which, as described earlier, can be heated in step 9 at elevated temperatures to the same 9 ⁇ ,10 ⁇ and 9 ⁇ ,10 ⁇ provitamin D diastereomers 5, respectively.
- Table 2 lists subgroups of analogs falling within the scope of the Group II.
- the analogs listed in Group II are represented by the analogs identified as JM, JN, JO and JP. These analogs, depending on their structure, have a biological activity as agonists or antagonists of slow genomic responses or the rapid
- the antagonists are represented by the generic formula II wherein Cl hydroxyl is in ⁇ configuration,
- C3 hydroxyl is in ⁇ configuration
- C9 hydrogen is in ⁇ and CIO methyl is in ⁇ configuration
- R x is the substituent II-l, II-2, II-4 and 11-10, preferably the substituents II-l, II-2, and 11-10, or wherein Cl hydrogen is in ⁇ and C3 is in ⁇ configuration
- C9 hydrogen is in ⁇ and CIO methyl is in ⁇ configuration
- R ⁇ is the substituent II-l, II-2, II-7, II- 10, and is preferably the substituent II-l, II-2 or 11-10.
- the agonists are represented by the generic formula II wherein Cl hydroxyl is in ⁇ configuration, C3 hydroxyl is in ⁇ configuration, C9 hydrogen is in ⁇ and CIO methyl is in ⁇ configuration and R x is the substituent II-l, II-2, II-4, 11-10, and preferably it is the substituent II-l, II-2, and 11-13.
- the specific agonist of this group is the analog JO where Ri is the substituent II-l. Preparation of the analog JO is described in Example 6.
- the other agonists of the Group II are represented by the general formula II wherein Cl hydroxyl is in ⁇ and C3 hydroxyl is in ⁇ configuration, C9 hydrogen is in ⁇ and CIO methyl is in ⁇ configuration and R x is the substituent II-l, II-2, II-4, 11-10 and, preferably, it is the substituent II-l, II-2 and 11-10.
- the specific agonist of this group is the analog JN where R x is the substituent II-l.
- the other agonists of the Group II are represented by the general formula II wherein Cl hydroxyl is in ⁇ and C3 hydroxyl is in ⁇ configuration, C9 hydrogen is in ⁇ and CIO methyl is in ⁇ configuration and R_ is the substituent II-l - 11-10, preferably the substituent II-l, II-2, II-4 and 11-10.
- the specific agonist of this group is the analog JM where R t is the substituent II-l. Preparation of the analog JM is described in Example 5.
- Still another agonists of the Group II are represented by the general formula II wherein Cl hydroxyl is in ⁇ and C3 hydroxyl is in ⁇ configuration, C9 hydrogen is in ⁇ and CIO methyl is in ⁇ configuration and R ⁇ is the substituent II-l - 11-10, preferably II-l, II-2 , II-4 and 11-10.
- the specific agonist of this group is the analog JP where R ⁇ is the substituent II-l.
- Preparation of the analog JP is described in Example 6.
- the group III is represented by compounds having a general formula III
- R_ is not the substituent III-l, III-2, III-3 , III-9 , 111-10; or when Cl and C3 hydroxyls are ⁇ - ⁇ and C14 hydrogen is ⁇ and C16-C17 is a single or double bond, then R : is not the substituent III-4 and III-5.
- R_ refers to the substituents of generic formula (III) or the suitably protected forms (R' D , usually as the silyl ether; all structures may have single or double bonds across the C16- 17 positions.
- Reaction Scheme 3 illustrates preparation of compounds of the Group III.
- Compounds of the general formula III are prepared according to Scheme 3 using the two general approaches shown as Scheme 3-A and Scheme 3-B.
- the starting A-ring fragments 1 and 5, wherein the C1-C3 alcohols are masked as their TBDMS ether protecting groups (chemical acronyms throughout this patent document follow the guidelines of the "Notice to Authors" of the Journal of Organic Chemistry) , as well as the CD fragments 2 and 6, wherein the substituent R ⁇ is the alcohol protected form of R x given in general formula II, are easily prepared according to references listed above.
- each compound may have a single or double bond across C16-C17.
- compound 4 is the same as the compound having general formula III.
- Reactions illustrated in Scheme 3-A begins with the palladiu (0) mediated coupling of 1 with 2 in step 1 to afford compound 3a. Deprotection of 3a in step 2 to gives compound 3b followed by Lindlar semi-hydrogenation (step 3) of the latter (3b) affords the desired previtamin type compound 4.
- step 3B the A-ring phosphine oxide 5 , after deprotonation in step 4 , is coupled with CD ring fragment 6 in step 5 followed by deprotection in step 6 with TBAF and THF.
- Table 3 lists subgroups of analogs falling within the scope of the Group III.
- the analogs listed in Group III are represented by the analog identified as GF. These analogs of Group III, depending on their structure and configuration, have a 25 biological activity as agonists or antagonists of slow genomic responses and agonists or antagonists of the rapid nongenomic responses.
- C14 hydrogen is in ⁇ or ⁇ configuration
- C16-C17 is a single or double bond
- R x is the substituent III-l - 111-10.
- Preferred group of compounds of the Group III are compounds wherein Cl is in ⁇ configuration, C3 is in ⁇ 35 configuration and the R x substituent is selected from the group III-l - 111-10.
- the specific agonist of this group is the analog GF where R ⁇ is the substituent III-l.
- Preparation of the analog GF is described in Example 2. 40
- the group IV is represented by compounds having a general formula IV
- R x is a substituent selected from the group consisting of substituents IV-1 through IV-10
- R refers to the substituents of generic formula (IV) or the suitably protected forms (R' ⁇ ) , usually as the silyl ether; all structures may have single or double bonds across the C16-C17 positions.
- R ⁇ refers to the substituents of generic formula (IV) or the suitably protected forms (R' ⁇ ), usually as the silyl ether; all structures may have single or double bonds across the C16-C17 positions.
- Reaction Scheme 4 illustrates preparation of compounds of the Group IV.
- Compounds of the general formula IV are prepared according to the general reaction Scheme 4.
- the starting A-ring fragment 1, wherein the C1-C3 alcohols are masked as their TBDMS ether protecting groups (chemical acronyms throughout this patent document follow the guidelines of the "Notice to Authors" of the Journal of Organic
- each compound may have a single or double bond across C16-C17.
- the vitamin D A-ring fragment 1 is treated in step 1 with butyllithium and then the resulting lithium salt is added to ketone 2 in step 2 .
- the resulting product is directly reacted in step 3 with butyllithium and then benzoyl chloride to afford the propargyl benzoate 3.
- reduction of 3 using samarium iodide, catalytic palladium(O) reagent, and isopropyl alcohol affords an intermediate allene in step 4 which is directly deprotected using TBAF and THF in step 5 to afford the 6 ⁇ - vinylallene analog 4.
- Photochemical irradiation as in step 6 using a 450 watt medium pressure mercury lamp with methanol as solvent affords the corresponding 6 ⁇ -diastereomer 5.
- the vinylallenes 4 and 5 are more generally represented by the generic structure IV.
- Table 4 lists subgroups of analogs falling within the scope of the Group IV.
- analogs listed in Group IV are represented by the analogs identified as analogs JV and J . These analogs, depending on their structure and configuration, have a biological activity as agonists of slow genomic responses or as agonists or antagonists of the rapid nongenomic responses.
- the agonists and antagonists are represented by the generic formula IV wherein Cl hydrogen is in ⁇ or ⁇ configuration, and C3 is in ⁇ or ⁇ configuration, C5-C6 is in ⁇ or ⁇ configuration, C14 hydrogen is ⁇ , C16-C17 is a single or double bond and R ⁇ is a substituent selected from the group consisting of substituents IV-1 through IV-10.
- Preferred agonists in this group of compounds of this group are compounds wherein Cl is in ⁇ configuration, C3 is in ⁇ configuration and the R 1 substituent is IV-1.
- the specific agonists of this group are the analogs JV and JW.
- Cl and C3 are positional isomers ⁇ and ⁇ which may be the same or different in ⁇ , ⁇ , ⁇ or ⁇ configuration, wherein C5-C6 double band is cis and C7C8 double band is trans; wherein C16-C17 is a single or double bond; and wherein R x is a substituent is selected from the group consisting of substituents V-l through V-10
- R 1 refers to the substituents of generic formula (V) or the suitably protected forms (R' ⁇ ) , usually as the silyl ether.
- R'i is the alcohol protected form of Ri given in general formula V, are prepared according to Chemical Reviews, 95: 1877-1952 (1995). J. Or ⁇ . Chem. f 58: 1895-1899 (1993); J__. Org. Chem., 54: 4072-4083 (1989) as cited above. Each compound may have a single or double bond across C16-C17. Thus, 5 is the same as generic structure V.
- Scheme 5-A starts with the palladium ⁇ 0) mediated coupling of 1 with 2 in step 1 to afford 3a, which in turn can be deprotected in step 2 using TBAF and THF to afford the free alcohol 3b.
- Lindlar catalyzed hydrogenation of 3b affords previtamin 4 which upon heating and refluxing isooctane as given in step 4 produces the desired analog 5.
- the A-ring phosphine oxide 6 is directly treated with strong base as shown in step 5 whereupon Horner-Wittig reaction with ketone 7 produces a protected triene as given in step 6.
- Deprotection of the resulting product with TBAF and tetrahydrofuran in step 7 of Scheme 5 also affords the same analog 5.
- analog LO which is an agonist of slow genomic and rapid nongenomic responses.
- Vitamin D is essential for maintenance of calcium/mineral homeostasis.
- One of the __ vitamin D metabolites, namely l ⁇ , 25 (OH) 2 -vitamin D 3 [l ⁇ , 25 (OH) 2 D 3 ] is a steroid hormone and therefore the number of the biological responses attributable to the parent vitamin D occur in a steroid hormone-like fashion through its metabolite l ⁇ ,25(OH) 2 D 3 .
- l ⁇ ,25(OH) 2 D 3 has additional multidisciplinary actions in tissues not primarily related to mineral metabolism, such as, for example, its effects on cell differentiation and proliferation including interaction with cancer cells detectable in leukemia, breast, prostate, colon tumor growth, the immune system, skin, selected brain cells, and its participation in the process of peptide hormone secretion exemplarized by parathyroid hormone or insulin.
- Figure 1 shows the vitamin D endocrine system and its core elements.
- the core elements of the vitamin D endocrine system include the skin, liver, kidney, blood circulation and other
- Vitamin D (7-dehydrocholesterol) to vitamin D 3 occurs in the skin. Vitamin D 3 is then metabolized by the liver to 25(OH)D 3 .
- the kidney functioning as an endocrine gland, converts 25(OH)D 3 to l ⁇ ,25(OH) 2 D 3 and
- vitamin D binding protein DBP
- Figure 2 presents a more comprehensive version of the vitamin D endocrine system specifically indicating selective generation of biological responses by the analogs of 20 l ⁇ ,25(OH) 2 D 3 resulting in the treatment of specif-ied disease states.
- a detailed tabulation of the cells containing the nuclear receptor [VDR nuc ] for l ⁇ ,25(OH) 2 D 3 as well as an enumeration of the tissue location of the membrane receptor [VDR mem ] where rapid response is initiated are seen in the 25 lower part of the Figure 2.
- Figure 2 additionally shows the target sites for application of l ⁇ , 25 (OH) 2 D 3 analogs functioning as agonist and antagonist.
- Vitamin D is a seco steroid, thus its 9,10 carbon-carbon bond is broken, and because it has an eight carbon side chain, both the parent vitamin D and all its metabolites and analogs are unusually conformationally flexible. Such conformational
- Figure 3 illustrates the conformational flexibility of vitamin D molecules using l ⁇ ,25(OH) 2 D 3 as an example.
- Figure 3A shows the dynamic single bond rotation of the cholesterol-like side chain of l ⁇ , 25 (OH) 2 D 3 , that has 360° rotations about five single carbon bonds and the oxygen as indicated by the curved arrows.
- the dots indicate the position in three-dimensional space of the 25-hydroxyl group for some 394 readily identifiable side chain conformations which have been determined from energy minimization calculations.
- Figure 3A Two orientations of the C/D side chain are seen in Figure 3A, a top view, and in Figure 3B, an in plane view.
- Figure 3B shows the rapid (thousands of times per second) chair-chair interconversion of the A-ring of the secosteroid which effectively equilibrates the l ⁇ -hydroxyl between the axial and equatorial orientations.
- Figure 3C shows the 360° rotation rotational freedom about the 6,7 carbon-carbon bond of the seco B-ring which generates conformations ranging from the more steroid-like (6-s-cis) conformation, to the open and extended (6-s-trans) conformation of l ⁇ , 25 (OH) 2 D 3 .
- Conformationally flexible analogs of l ⁇ ,25(OH) 2 D 3 as seen in Figure 3 can interact with both the VDR nuc and the VDR, relieve em while 6-s-cis locked conformationally restricted analogs interact only with the VDR_ em .
- a tabulation of the analogs of the invention, their conformational flexibility and general biological properties are presented in Table 6.
- Vitamin D binding protein is an important part of the system utilized for the delivery of the vitamin D, its metabolites or its analogs to the target organs.
- the key role played by its metabolites the DBP in transporting both l ⁇ ,25(OH) 2 D 3 and its analogs, both conformationally flexible and conformationally restricted, throughout the physiological system is shown in Figure 4.
- Figure 4 is schematic model of the role of the vitamin D-binding protein (DBP) in transporting l ⁇ ,25(OH) 2 D 3 or its analogs throughout the circulatory system.
- DBP vitamin D-binding protein
- DBP either binds l ⁇ ,25(OH) 2 D 3 as it is secreted by the kidney or binds analogs at their site of the encounter following the analog administration.
- the DBP binds it after its intestinal absorption.
- DBP binds to the venously administered and available analog in the circulating blood.
- the relatively water insoluble analogs would not find their way in the body to the site of target cells, which are, by definition, any cells in the body that possess either membrane receptors (VD ⁇ ,,) or nuclear receptors (VDR flank UC ) for l ⁇ ,25(OH) 2 D 3 .
- the DBP bound to the analog moves through the circulatory system and makes the bound analog universally available throughout the circulatory system to all cells that are subserved.
- the DBP has a specific ligand binding domain created via its protein secondary structure.
- the DBP ligand has a different ligand specificity from that of the VDR literal UC and VDR. ⁇ receptor ligand binding domains, seen in Figures 4 and 5.
- the analogs are bound noncovalently by the DBP ligand. Accordingly, there is a continual binding and release of l ⁇ ,25(OH) 2 D 3 or analogs governed by the equilibrium constant or affinity for ligand binding by DBP. The important consequence is that there are low 5 concentrations of free analogs distributed throughout the circulatory system which are available for uptake by target cells and interaction with the VDR formulate UC and/or VDR M ,.
- the DBP has the capability to transport the conformationally flexible l ⁇ ,25(OH) 2 D 3 , conformationally flexible analogs and 6-s-cis conformationally restricted analogs.
- the spectrum of biological responses mediated by the hormone l ⁇ ,25(OH) 2 D 3 occurs as a consequence of the interaction of l ⁇ ,25(OH) 2 D 3 with two classes of specific receptors. These receptors are identified as the nuclear receptor, VDR nuc and the cellular membrane receptor, VDR mem .
- the VDR nuc protein contains a ligand binding domain able to bind with high affinity and with great specificity l ⁇ ,25(OH) 2 D 3 and closely related analogs.
- l ⁇ ,25(OH) 2 D 3 has been found to generate biological responses via interaction with a putative membrane receptor [VDR raem ] which is coupled to cellular signal transduction pathways. This interaction generates rapid response via opening voltage gated Ca 2+ channels and Cl " channels as well as activating
- MAP-kinases Different shapes of the conformationally flexible l ⁇ ,25(OH) 2 D 3 or its analogs bind to the VDR nuc and VO ⁇ and initiate biological responses via activation of signal transduction mechanisms which are coupled to either the VDR nuo or the VD ⁇ em * Thus the totality of biological responses mediated by l ⁇ ,25(OH) 2 D 3 or its analogs represents an integration of both nuclear receptor and membrane receptor initiated events.
- analogs of l ⁇ ,25 (OH) 2 D 3 there are two general classes of such analogs. There are agonists that generate responses similar to l ⁇ ,25(OH) 2 D 3 and there are antagonists that block or minimize the responses initiated by l ⁇ ,25(OH) 2 D 3 or agonist analogs. Further, agonist or antagonist molecules can either be fully conformationally flexible, like the natural hormone l ⁇ ,25(OH) 2 D 3 as seen in Figure 3, or be conformationally restricted.
- a conformationally restricted agonist molecule is l ⁇ ,25(0H) 2 -7-dehydrocholesterol, analog JM, that is permanently locked in the 6-s-cis shape.
- One class are those analogs that have complete flexibility around the 6,7 carbon-carbon bond, as does l ⁇ ,25(OH) 2 D 3 .
- the second class are those analogs which are conformationally restricted, such as 6,7-locked analogs.
- An example of such analogs are l ⁇ ,25(OH) 2 -7-dehydrocholesterol (JM) or l ⁇ ,25(OH) 2 -lumisterol (JN) .
- Figure 5 compares the mode of actions of these two types of analogs, namely conformationally flexible analogs and conformationally restricted 6-s-cis analogs.
- l ⁇ ,25(OH) 2 D 3 which is conformationally flexible interacts with both the membrane receptor depicted as VDR mem located in the cell membrane, and with the cell nuclear receptor depicted as VDR- UC located in the cell nucleus of the target cell.
- the slow genomic responses appear after l ⁇ ,25(OH) 2 D 3 or its analog's interaction with VDR nuc . Rapid responses are generated upon interaction of l ⁇ ,25(OH) 2 D 3 or its analog with VD mem .
- Conformationally flexible analogs of the invention act similarly to l ⁇ ,25(OH) 2 D 3 generating the same general biological responses as those illustrated in Figure 5A, i.e., both slow and rapid responses as a consequence of interacting with both VDR nuc and VD m ⁇ _.
- Figure 6 represents a model and a description of the mechanisms of action by which l ⁇ ,25(OH) 2 D 3 generates biological responses in target cells.
- the conformationally flexible natural hormone, l ⁇ ,25 (OH) 2 D 3 , and conformationally flexible analogs interact with both the VDR nuc and VD mem .
- 6-s-cis locked analogs can interact only with the VD mem .
- the bottom panel of the Figure 6 lists certain target cells for l ⁇ ,25(OH) 2 D 3 and identifies typical responses of these cells to administration of l ⁇ ,25(OH) 2 D 3 or the analog which occur there. Disease states for treatment with analogs of l ⁇ ,25(OH) 2 D 3 are listed in Figure 6 bottom.
- the right side of Figure 6 describes the mechanism of action for ligands, both conformationally flexible and 6-s- ⁇ is locked analogs, that bind to the VD mem to initiate the generation of rapid biological responses. Occupancy of the VDR mem can lead to activation of a variety of intracellular messengers, such as cyclic AMP, protein kinase C, or increases in intracellular Ca 2+ concentration, which, depending upon the cell type, can cause the opening of calcium channels, chloride channels, or activation of mitogen-activated protein kinase.
- intracellular messengers such as cyclic AMP, protein kinase C
- Ca 2+ concentration which, depending upon the cell type, can cause the opening of calcium channels, chloride channels, or activation of mitogen-activated protein kinase.
- the MAP-kinase activation leads to changes in the phosphorylation state of the proteins participating in the transcription complex, including the VDR nuc . Then, depending upon whether the gene subject to regulation by the VDR nuc is subject to up-regulation or down-regulation, there can be 0 further modulation of this process so that the final outcome of the slow genomic response is favorably enhanced.
- the details of the enhancement is dependent upon the cell type in which the
- MAP-kinase was activated.
- the bottom portion of Figure 5 links integration of rapid and slow genomic signal transduction 5 processes to the overall outcome biological response for a variety of target cells.
- dysfunction of the signal transduction process in the designated target cells can lead to the onset of a variety of disease states as seen in Figure 5, bottom right column. 0 III.
- Agonists (a) Conformationally Flexible Genomic Agonist Analogs are the 5 analogs which interact with the nuclear receptor for l ⁇ ,25(OH) 2 D 3 VDR nuc and are, therefore, involved in the slow genomic responses. Exemplary analogs in this group are analogs listed in Table 7.
- Conformationally restricted genomic agonist analogs are the analogs which bind with a specificity to the vitamin D nuclear receptor VDR nuc and are therefore also involved in genomic responses .
- Conformationally flexible antagonist of genomic responses function as antagonists of the vitamin D nuclear receptor.
- Conformationally Restricted Antagonists ⁇ £ Ra&id
- Conformationally restricted antagonists of rapid responses function as antagonists of the VDR mem -
- Analog utility and its activity is dependent on its binding to the vitamin D-binding protein (DBP) . Only if the analog is able to bind to the DBP can it be delivered to the target organ.
- DBP vitamin D-binding protein
- Analog binding to the DBP is illustrated in Figure 4 which summarizes the key role played by the vitamin D binding protein in the transport of l ⁇ ,25(OH) 2 D 3 or its analogs through the blood compartment, from its site of administration or uptake to make them available for uptake by target cells.
- the vitamin D-binding protein is a protein of about 50 kDa containing a ligand binding domain which can recognize and discriminate various functional groups and structural modifications on potential ligands, i.e. analogs of l ⁇ ,25(OH) 2 D 3 . Since DBP determines the availability of its bound ligand to target cells, it is important to define the relative affinity of a given analog to bind to DBP. The affinity of binding of the analog to the DBP binding site is measured and expressed as Relative Competitive Index.
- Figure 7 shows results of the determination of the RCI for representative analogs for the vitamin D binding protein (DBP) compared to l ⁇ ,25(OH) 2 D 3 , identified as compound C.
- the compared analogs are 14 ⁇ , 15 ⁇ -methano-l ⁇ , 25 (OH) 2 D 3 (LO) , 22 (m(dimethylhydroxymethyl) phenyl) -23 ,24,25,26, 27-pentanor-l ⁇ - 0H-D 3 (EV) and (22R)-l,25(OH) 2 -22,23,diene-D 3 (HR) , all conformationally flexible genomic agonists.
- the RCI values expressed as (% maximum bound) "1 x 100 of the analog in competition with l,25(OH) 2 D 3 are indicated in the Figure 7.
- the RCI for l ⁇ ,25(OH) 2 D 3 is set to 100%.
- the data seen in Figure 7 represent the mean of three determinations.
- analog JX has the highest RCI for DBP, a value of 211,000 or 2110 times greater than the reference l ⁇ ,25(OH) 2 D 3 .
- This analog therefore, binds very tightly to DBP and has a much lower free concentration and lower availability for uptake by target cells.
- analog HL has an RCI of only 0.1, which is 1000 times lower than that of the reference l ⁇ ,25 (OH) 2 D 3 .
- this analog binds poorly to DBP and has a much higher free concentration and, therefore, a higher availability for uptake by target cells if brought to their vicinity.
- Table 11 summarizes the biological evaluation of all the analogs of l ⁇ ,25(OH) 2 D 3 which are subject of this invention.
- Table 11 identifies biological properties, such as genomic response, rapid response, agonist or antagonist function, binding of the analog to the vitamin binding protein (expressed as RCI), binding to the nuclear l ⁇ ,25(OH) 2 D 3 receptor (expressed as RCI) rapid response (expressed as % transcaltachia the rapid hormonal stimulation of intestinal calcium absorption) the classic vitamin D responses such as intestinal Ca 2+ absorption (ICA) and bone Ca 2+ mobilizing activity (BCM) determined in vivo in a vitamin D-deficient chick, and cell differentiation (expressed as % ED50) , an assessment of the ability to promote the nuclear response of cell differentiation.
- RCI vitamin binding protein
- RCI nuclear l ⁇ ,25(OH) 2 D 3 receptor
- RCI rapid response
- the classic vitamin D responses such as intestinal Ca 2+ absorption (ICA) and bone Ca 2+ mobilizing activity (BCM) determined in vivo in a vitamin D-deficient chick
- ICA intestinal Ca 2+ absorption
- BCM bone Ca 2+ mobilizing activity
- analogs 22 As seen in Table 11, twenty three analogs and l ⁇ ,25(OH) 2 D 3 (designated by analog code as C) were submitted to testing as outlined in Table 6.
- analogs 22 are agonists, that is compounds which possess affinity for the receptor and are capable of combining with l ⁇ ,25(OH) 2 D 3 receptor.
- One of the analogs is an antagonist (HL) , that is a compound which does not bind to the receptor and in fact it blocks or inhibits the action of agonist for rapid responses.
- the best analog identified by its comparative activity with l ⁇ ,25(OH) 2 D 3 is the analog LO, showing 30% of ICA and 50% of BCM, compared to l ⁇ ,25(OH) 2 D 3 .
- the l ⁇ ,25(OH) 2 D 3( or the analog of the invention After transport and delivery of l ⁇ ,25(OH) 2 D 3( or the analog of the invention by DBP through the circulatory system, the l ⁇ ,25(OH) 2 D 3 or the analog is disassociated from the DBP. The l ⁇ ,25 (OH) 2 D 3 , or the analog, then diffuses as free molecule through the extracellular fluid to come into very close proximity of a target cell.
- the target cell by definition, is a cell possessing either or both the VDR nuc and VDR ra ⁇ m .
- panel A the conformationally flexible l ⁇ ,25(OH) 2 D 3 , or the analog, then interacts either directly with the VDR m ⁇ m present on the outer cell membrane or, alternatively, diffuses through the outer cell membrane and enters into the cytosol or soluble portion of the cell where it encounters and interacts with the VDR nUC .
- a very tight receptor ligand complex is formed virtually exclusively in the nuclear portion of the cell.
- Resident in the nucleus of the cell is the DNA that comprise all the genes that describe the blueprints for that given organism (see Figure 6, left side) .
- the genetic information inherent in the DNA of the given gene is utilized via initiation of a complex process known as transcription and translation.
- the transcription process involves conversion of the information resident in the sequence of nucleotides comprising the DNA into messenger RNA molecules.
- the process of translation then describes the biological processes wherein the mRNA molecules are translated by the process of protein biosynthesis to result in the production of protein molecules.
- VDR- UC will search out amongst all the DNA resident in the nucleus, those genes which have incorporated into them the so-called vitamin D response element (VDRE) .
- VDRE vitamin D response element
- the transcription complex is comprised of the DNA of a specific gene that contains a VDRE and, as well, other protein enzymes that are necessary to convert the blueprint information of the DNA into the generation of new messenger RNA molecules.
- VDRE There are two general categories of VDRE. One category comprises those that result in stimulation of the transcription process , that is an increase in the number of mRNA molecules that are produced. Another category comprises those which repress, that is reduce the number of mRNA molecules that are produced.
- FIG 8 shows Relative Competitive Index (RCI) determination for representative analogs that bind to the nuclear receptor for l ⁇ ,25(OH) 2 D 3 [VDR nuc ] .
- the assay is based upon the principles of a steroid competition assay. A fixed amount of [ 3 H]l ⁇ ,25 (OH) 2 D 3 is mixed with increasing amounts of competitive analogs or the natural hormone, l ⁇ ,25 (OH) 2 D 3 , and incubated with a VDR nUC receptor preparation from chick intestine ucosa. The results are presented for l ⁇ ,25 (OH) 2 D 3 , analog LO
- Bone calcium Mobilization Assays A primary fundamental physiological property of vitamin D and particularly l ⁇ ,25(OH) 2 D 3 is its ability to stimulate the intestinal absorption of calcium and facilitate the availability of dietary calcium to the organism. Intestinal absorption of the calcium is measured by the intestinal calcium absorption (ICA) assay, developed in the model of vitamin-D deficient chicks. The ICA assay was used to determine the relative capability of the tested analog to stimulate intestinal Ca 2+ absorption.
- ICA intestinal calcium absorption
- a second important physiological action of l ⁇ ,25(OH) 2 D 3 is its effects on bone cells. Under circumstances of a dietary shortage of calcium, the blood concentration of Ca 2+ falls and the individual becomes hypocalcemic. In order to prevent an extreme reduction in the blood concentration of Ca 2+ , the organism utilizes l ⁇ ,25(OH) 2 D 3 to activate bone resorbing cells, the osteoclasts, which in turn mobilize bone calcium and contribute it to the blood calcium pool thereby alleviating the hypocalcemia.
- the bone calcium mobilizing (BCM) assay is also conducted in the vitamin D-deficient chick.
- the BCM assay determines the relative ability of the tested analog to mobilize bone calcium.
- the natural hormone l ⁇ ,25(OH) 2 D 3 is very potent in the BCM assay. For example, when l ⁇ ,25(OH) 2 D 3 in inappropriate amounts are used as a drug in human patients, the patient may become hypercalcemic and eventually hypercalciuria with nephrolithiasis and renal failure may develop.
- the BCM assay was used to determine the relative activity of the analogs of the invention to stimulate bone Ca 2+ mobilization.
- Results are expressed as mean ⁇ SE of groups of seven chicks. Each assay included a negative control (-D) , that is no vitamin D was present, and a positive control, where vitamin D 3 (+D 3 ) was present in 3.25 nmol. The difference between the -D and +D 3 groups was significant at P ⁇ 0.01. l ⁇ ,25(OH) 2 D 3 and analogs LO [14 ⁇ ,15 ⁇ - methano-l ⁇ ,25(OH) 2 D 3 ] , EV [22- (m(dimethylhydroxymethyl) phenyl) -
- Table 6 shows ICA and BCM data for the analogs seen in Figure 9 as well as other analogs of the invention.
- analog LO which has the highest ICA (30%) and BCM (80%) relative to the ICA and BCM values for l ⁇ ,25(OH) 2 D 3 would be a highly effective stimulator of bone Ca 2+ mobilizing activity (BCM) and reasonable stimulator of intestinal Ca 2+ absorption (ICA) and is therefore useful for treatment of hypocalcemia and rickets.
- analogs DE and EV show stimulating activity in both ICA and BCM assays.
- l ⁇ ,25(OH) 2 D 3 One of the recently discovered properties of the natural hormone l ⁇ ,25(OH) 2 D 3 , in addition to its involvement in calcium metabolism, is its potent ability to promote cell differentiation and/or inhibit cell proliferation, both these activities are related to cancer. These actions of l ⁇ ,25(OH) 2 D 3 are dependent upon the widespread tissue distribution of receptors, both the VDR nuc and VDR mem , as described in Figure 2. l ⁇ ,25(OH) 2 D 3 has been shown to be a potent cell differentiating agent in a variety of cell lines related to pathological states, such as leukemia, breast cancer, prostate cancer, and colon cancer, and as well in keratinocytes, cartilage cells, bone forming osteoblasts and the immune system cells.
- the cell differentiation assay is used for a determination of relative potency of the analog vis-a-vis the potency of the reference compound l ⁇ ,25(OH) 2 D 3 in promoting the cell differentiation or inhibiting the cell proliferation.
- the results of the cell differentiation assay are expressed as the effective dose-50 (ED-50) which is defined as 50% of the concentration required for a maximal response.
- ED-50 of l ⁇ ,25(OH) 2 D 3 is determined to be 1. If the analog has ED-50 of 0.1, it achieves 50% of its maximal cell differentiation effect at a concentration of about one tenth that of l ⁇ ,25(OH) 2 D 3 and is, therefore, ten times more effective.
- Figure 10 is dose-response of analog HS or l ⁇ ,25(OH) 2 D 3 on differentiation of HL-60 cells. The results are expressed as a percentage of untreated HL-60 cells which acquired, as a consequence of cell differentiation, the ability to effect reduction of nitro blue tetrazolium (NBT) . Each point represents the mean of two experiments with triplicate dishes. Open circles (0) show l ⁇ ,25(OH) 2 D 3 ; closed circles (•) show analog HS.
- analog HS is significantly more potent than l ⁇ ,25(OH) 2 D 3 in promoting the cell differentiation of HL-60 cells.
- Analog HS was found to have an ED-50 of 0.05 as compared to the 1.00 for l ⁇ ,25(OH) 2 D 3 and is therefore about twenty times more potent at promoting the cell differentiation of HL-60 cells.
- Rapid responses are initiated by occupancy of the VDR me ,,, with an analog ligand that has the shape of a 6-s-cis oriented l ⁇ ,25(OH) 2 D 3 . Rapid responses of the analogs of the invention are detected by their ability to achieve transcaltachia or mitogen activated protein kinase.
- Transcaltachia Transcaltachia is defined as the rapid stimulation of calcium transport across an epithelial cell of a perfused intestine. The process of transcaltachia is stimulated by hormone D [l ⁇ ,25(OH) 2 D 3 ] or, according to the current invention, by 6-s-cis conformationally restricted analogs.
- transcaltachia is a rapid response which occurs within one to several seconds to up to about three minutes as compared to a genomic response which is slow and usually takes about several minutes to several hours. The events comprising the initiation of the rapid response of transcaltachia by 6-s-cis conformationally restricted analogs are described below.
- Transcaltachia is a component of the overall process describing the intestinal absorption of calcium, which is the classic response related to the vitamin D.
- vitamin D is essential because it increases the uptake of dietary calcium and makes it available for incorporation into the bones.
- the active agent of vitamin D 3 that is responsible for the stimulation of intestinal calcium absorption is a vitamin D metabolite l ⁇ ,25 (OH) 2 D 3 , also called hormone D.
- the general process of calcium transport across an intestinal epithelial cell involves three steps.
- the first step is the ingestion of calcium from food and the movement of calcium into the lumen of the intestine. Once the calcium is present in the small intestine, it moves across the outer brush- border membrane of the cell and into the interior of the epithelial cell.
- the second step is the calcium accumulation in membrane bounded vesicles known as lysosome-like vesicles. These calcium-bearing vesicles then move across the interior of the cell and respond to a signal indicating that they should be exported out of the cell into the adjacent blood compartment.
- the third step involves an initiating signal for the export of calcium out of the cell (exocytosis) regulated by hormone D in a 6-s- ⁇ is shape or by 6-s- ⁇ is locked analogs of the invention which are delivered by vitamin D binding protein (DBP) to the exterior surface of the epithelial cell.
- DBP vitamin D binding protein
- the hormone D or the 6-s-cis locked analog is unloaded from the DBP in its free form immediately adjacent to the outer cell membrane of an epithelial cell where the receptor VO - em is resident, as shown in Figure 4.
- the VDR mem i specific only for compounds in the 6- s-cis orientation and therefore binds only hormone D or analogs of hormone D which are in the 6-s-cis locked shape.
- Formation of the receptor bound ligand complex that is a VDR mem /6 ⁇ s-cis analog, results in the generation of a biological signal involving opening of voltage-gated calcium channels that send a message to the interior of the cell so that there is a prompt (rapid) initiation of the export of the calcium bearing lysosomal-like vesicles.
- this activity is identified as a rapid response.
- This export process occurs within 1-3 minutes.
- the net effect of the delivery of a 6-s- ⁇ is locked analog by DBP to the blood bathed surface of an intestinal epithelial cell is the prompt stimulation of intestinal calcium transport that results in an increased exiting of calcium from the interior of the epithelial cell into the blood compartment.
- FIG. 11 is illustrative of the rapid response of transcaltachia and shows the effectiveness of conformationally restricted analogs JN and JM to stimulate the rapid response of transcaltachia.
- the reference compound is the conformationally flexible l ⁇ ,25 (OH) 2 D 3 , which is able to achieve the shape of the 6-s-cis locked conformationally restricted analogs and thus interact with the VDR mem which has been implicated in transcaltachia.
- Findings that only 6-s-cis locked analogs can elicit transcaltachia is extremely important for their therapeutic utility. While l ⁇ ,25(OH) 2 D 3 has general utility for both genomic and rapid responses and is, therefore, much less specific, by identifying only certain types of analogs, that is 6-s-cis locked analogs as being able to elicit transcaltachia, the treatment of osteoporosis, for example, can be achieved without danger of causing hypercalcemia which can happen if large doses of l ⁇ ,25(OH) 2 D 3 are administered. Such doses inappropriately activate the bone resorbing cells or osteoclasts.
- Figure 11 represents stimulated 45 Ca 2+ transport in duodenal loops vascularly perfused with l ⁇ ,25(OH) 2 D 3 or l ⁇ ,25(OH) 2 -7-dehydrocholesterol (JM) , or l ⁇ ,25(OH) 2 -lumisterol (JN) .
- Duodenal loops from normal, vitamin D-replete chicks were lumenally perfused with 45 Ca 2+ (5 uCi/ml of buffer) .
- celiac artery of controls were perfused with control medium for the first 20 min.
- duodena were then either re-exposed to control medium containing the vehicle ethanol (0.005%, final concentration) through the celiac artery, or vascularly perfused with 300 pM or 650 pM agonist analogs JM or JN or with 650 pM of a control reference compound l ⁇ ,25(OH) 2 D 3 .
- the venous effluent was collected at 2 min intervals for liquid scintillation spectrophotometry of the 5 45 Ca 2+ .
- Figure 11A shows results obtained after perfusion with analog JM.
- Figure 11B shows results obtained after perfusion 0 with analog JN. Included in each graph are both the vehicle control and 650 pM l ⁇ ,25(OH) 2 D 3 of reference compound as a positive control.
- the results seen in Figures 11A and 11B indicate that the 6-s-cis locked analogs JM and JN are potent analogs of the rapid response process of transcaltachia. As 5 seen in these figures, within first four minutes, both analogs have activity comparable or better than the reference compound.
- analog JM has 60% of the potency of the conformationally flexible l ⁇ ,25(OH) 2 D 3 to stimulate transcaltachia, while analog JN is 105% as potent as 0 l ⁇ ,25(OH) 2 D 3 .
- analog JB [1 ( , 25 (OH) -tachysterol 3 ] was found to have smaller than 5% activity of l ⁇ ,25(OH) 2 D 3 in stimulating transcaltachia.
- Enzyme mitogen activated protein (MAP) kinase belongs to the family of serine/threonine protein kinases which can be activated by phosphorylation of a tyrosine residue induced by 0mitogens or cell differentiating agents. MAP-kinase integrates multiple intracellular signals transmitted by various second messengers, and regulates many cellular functions by phosphorylation of several cytoplasmic kinases and nuclear transcription factors. 5 Agonists and antagonists of the invention activate or inhibit enzyme MAP-kinase localized in cytosolic/cell membranes and activate or inhibit related signal transduction pathways involved in modification of genomic responses of cells, for example, including their differentiation and/or proliferation.
- l ⁇ ,25-dihydroxyvitamin D 3 and particularly its 6-s-cis analogs are selective agonists of cytosolic localized mitogen- activated protein (MAP) -kinases. Further, l ⁇ ,25- dihydroxyvitamin D 3 (analog HL) is an antagonist of activation of MAP-kinases.
- NB4 human acute promyelocytic leukemia cells
- Figure 12A and 12B present the results of a densitometric scan of the Western blot analysis.
- the NB4 cells were treated with l ⁇ ,25(OH) 2 D 3 at 10 "8 M for 5 min and then extracted as described in Example 7.
- the lysate was further processed for anti-phosphotyrosine im unoprecipitation.
- the tyrosine- phosphorylated proteins were analyzed by Western blot according to Example 8. After transferring the proteins to the PVDF membrane, the membrane was further incubated with primary anti- p42 mapk antibodies that were (+) or were not (-) pre-exposed to
- Figure 12A shows results of a dose response by l ⁇ ,25(OH) 2 D 3 for activation of MAP-kinase at either 1 or 5 minutes exposure to it.
- l ⁇ ,25(OH) 2 D 3 significantly increased phosphorylation of p42 mapk in NB4 cells.
- the specificity of the immunodetected MAP-kinase was confirmed by pre-blocking of the primary anti-MAP-kinase antibody with purified MAP-kinase peptide in a Western blot step.
- Figure 12B presents results describing the ability of the conformationally flexible l ⁇ ,25(OH) 2 D 3 and a 6-s- ⁇ is locked analogs HF and JN to stimulate MAP-kinase activity in the human leukemia NB4 cell line. Testing conditions were the same as in Figure 12A. As seen in Figure 12B, analogs HF and JN activated MAP-kinase in 1 minute more than l ⁇ ,25(OH) 2 D 3 and were only slightly less active at 5 minute intervals.
- Antagonist Analogs A. Genomic Antagonists Genomic antagonists are compounds that function as antagonists of the vitamin D nuclear receptor. The genomic antagonists are believed to cause the VDR nuc to assume a conformation which blocks transcriptional machinery. B.
- Rapid response antagonists are compounds that function to antagonize the DVR mera .
- One representative conformationally flexible genomic antagonist is analog HL, namely l ⁇ ,25 (OH) 2 D 3 .
- Figure 13 illustrates the ability of 1B,25(0H) 2 D 3 to inhibit the agonist actions of l ⁇ ,25(OH) 2 D 3 on the rapid response of transcaltachia.
- the 1B,25(0H) 2 D 3 analog HL was added to the perfused duodenum either in advance or simultaneously with l ⁇ ,25(OH) 2 D 3 at varying concentrations.
- the data shown in Figure 13 are the mean ⁇ SEM from 4-5 duodena. Solid squares represent a combination of HL analog and l ⁇ ,25 (OH) 2 D 3 . Open circles represent the negative control receiving no treatment with l ⁇ ,25(OH) 2 D 3 or analog.
- Figure 13B shows the dose-response relationship of l ⁇ ,25(OH) 2 D 3 inhibiting the stimulation of transcaltachia by 300 pM l ⁇ , 25 (OH) 2 D 3 .
- Data represent the ratio of treated to basal values + SEM extracted from a time-course plot (as in panel A) at 32 minutes.
- transcaltachia caused by l ⁇ ,25(OH) 2 D 3 was particularly observable in Figure 13A-1 where the antagonist HL was tested at 12 pM in combination with l ⁇ ,25(OH) 2 D 3 at 300 pM.
- the antagonist was added at 60pM in advance of 300pM l ⁇ ,25(OH) 2 D 3 there was clear inhibition of transcaltachia ( Figure 13A-2) .
- a similar inhibition of transcaltachia occurred ( Figure 13A-3) when the antagonist was 300pM in advance of 300pM l ⁇ ,25(OH) 2 D 3 .
- transcaltachia When the antagonist was added at 400 pM and the l ⁇ ,25(OH) 2 D 3 was 300 pM, transcaltachia was clearly inhibited, as seen in Figure 13A-4. When the analog was administered before the transcaltachia, followed by the administration of l ⁇ ,25(OH) 2 D 3 , transcaltachia was almost completely inhibited and the transport of the calcium ion across the intestinal wall was inhibited.
- Utility of l ⁇ ,25(OH) 2 D 3 and other antagonist is based on their ability to inhibit the normal rapid actions of l ⁇ ,25(OH) 2 D 3 or other agonist and to block the intestinal absorption of calcium when the individual has an abnormally elevated blood concentration of Ca 2+ in blood.
- Antagonists of the invention are, therefore, useful for treatment of conditions such as hypercalcemia. They prevent exacerbation of the extant condition of hypercalcemia.
- analog l ⁇ ,25(OH) 2 D 3 has also been found to be capable of antagonizing rapid responses of l ⁇ ,25(OH) 2 D 3 to stimulate the opening of chloride channels in ROS 17/2.8 cells in osteoblast cells and the activation of MAP- kinase in human leukemia cells.
- Analog's HL antagonist action is illustrated by its ability to inhibit the rapid responses of l ⁇ ,25(OH) 2 D 3 . These antagonist actions are illustrated in Figures 14 and 15.
- Figure 14 shows opening or modulation of chloride channels in osteoblastic ROS 17/2.8 cells, following stimulation by l ⁇ ,25(OH) 2 D 3 .
- Figure 4 shows fold increase of outward currents in ROS 17/2.8 cells mediated by l ⁇ ,25(OH) 2 D 3 in the absence and presence of 1 nM l ⁇ ,25(OH) 2 D 3 .
- Fold increase of current amplitudes promoted by different concentrations of l ⁇ ,25(OH) 2 D 3 were measured for currents elicited by a depolarizing step to 80 mV, in the absence and presence of 1 nM HL in the bath.
- Figure 15 illustrates the stimulation of activation of MAP- kinase, specifically stimulation of phosphorylation of MAP- kinase by l ⁇ ,25-dihydroxyvitamin D 3 in promyelocytic NB4 leukemia cells.
- Figure 15 shows the effect of analog HL on l ⁇ ,25 (OH) 2 D 3 - induced p42 mapk phosphorylation in NB4 cells.
- A NB4 cells were treated with different doses of l ⁇ ,25(OH) 2 D 3 in the presence or absence of HL at 10 "9 M for 5 min.
- B Equal loading of total
- the analog HL is, therefore, useful for treatment of any disease which involves opening or closing calcium channels and stimulation of MAP-kinase. This would include the calcium absorption process, transcaltachia occurring in the intestine as well as the changes in chloride currents of the bone osteoblast (bone forming) cells. VIII. Therapeutic Utility of The Analogs of the Invention A. Evaluation of Therapeutic Utility of the Analyses.
- Analog's profile evaluation includes as the first step, its evaluation of its ability to interact with the VDR nuc and DBP binding proteins under in vitro steroid competition assays, as outlined in Figures 7 and 8.
- ICA intestinal Ca 2+ absorption
- BCM bone Ca 2+ mobilizing activity
- an appropriate cell culture or in vivo assay is conducted. This allows determination of the ability of the analog to achieve a favorable response in an animal model of the human disease state under study.
- the toxicology of in vivo chronic dosing with respect to the hypercalcemia-toxicity assay listed in the bottom line of Table 7, is performed and the analog is evaluated for its potential therapeutic activity.
- Table 12 presents a summary of animal models that have shown a demonstrated utility for drug development studies in the vitamin D endocrine system.
- Vitamin D Biochemical. Chemical and Clinical Aspects Related to Calcium Metabol sm, pp. 587-589, Berlin:
- the analog delivery to the target tissue is a primary aspect of the analog therapeutic utility. If the analog can be delivered to the target tissue quantitatively, then its therapeutic potential is high. If it cannot be delivered, then its therapeutic value is low.
- DBP vitamin D-binding protein
- the DBP is a protein of 50 kDa with a ligand binding domain which can recognize and discriminate various functional groups and structural modifications on potential ligands. As shown in Figures 7 and 8, the DBP recognizes and bind various analogs of l ,25(OH) 2 D 3 , which are subject of this invention with specific affinity. Since DBP determines the availability of its bound analog to target cells, it is important to define the relative affinity of a given analog to bind to DBP and also its ability to readily disassociate from such binding. The more available the analog is for uptake by a target cell, the more likely it is to interact with either the VDR- UC or the VDR- em and assert its therapeutic potential.
- compositions comprising conformationally flexible analogs or 6- s-cis locked analogs which are agonists or antagonists are used in administration modes as described in the following separate section of pharmaceutical compositions and modes of administration.
- Conformationally flexible analogs subject to this invention which are listed above in Table 2 are exemplarized by analogs 14 , 15 -me t h an o - l ⁇ , 25 ( OH ) 2 D 3 (LO) , 22- (m(dimethylhydroxymethyl)phenyl-23 , 14 , 15 , 16 , 17-pentan ⁇ r l (OH) D 3 (EV) , or l ⁇ ,18,25(OH) 3 D 3 (HS) .
- 6-s- ⁇ is locked analogs of l ⁇ ,25(OH) 2 D 3 subject to this invention which are listed above in Table 4 are exe plarized by analog l ⁇ ,25(OH) 2 -lumisterol (JN)
- exemplary and other listed analogs are useful for treatment of, among others, osteoporosis, osteomalacia, rickets, renal osteodystrophy, psoriasis, organ transplantation, and several cancers, such as leukemia and prostate cancer. All these diseases are caused by the vitamin D or its metabolites deficiency or may be corrected by treatment with vitamin D metabolites, particularly l ⁇ ,25(OH) 2 D 3 .
- Osteoporosis is the most common generalized disorder of bone characterized as a state of insufficiently calcified bone occurring as a consequence of a number of extraneous factors such as aging, menopause or other endocrine or nutritional deficiency. Due to these factors, the remodeling rate of bone is disturbed and there occurs either an increase in the relative rate of bone resorption or a decrease in the rate of bone formation.
- any analog able to elicit transcaltachia and which is responsive in classic intestinal absorption assay and bone calcium mobilization assay are good candidates for replacement of l ⁇ ,25(OH) 2 D 3 and for treatment and prevention of osteoporosis.
- Particularly active for treatment of osteoporosis are the drug formulations of the l ⁇ ,25 (OH) 2 D 3 , such as the conformationally flexible analogs LO [14, 15-methano- l ⁇ , 25 (OH) 2 D 3 ] , EV [22- (m(dimethylhydroxymethyl) phenyl- 23,14,15,16,17-pentanor l ⁇ (OH)D 3 ], or HS [l ⁇ , 18, 25 (OH) 3 D 3 ] or the drug formulations of 6-s-cis locked analogs of l ⁇ ,25(OH) 2 D 3 , such as analog JN [l ⁇ ,25 (OH) 2 -lumisterol] .
- These drugs are used to treat those forms of osteoporosis which are related to a lowered level of serum l ⁇ , 25 (OH) 2 D 3 , because they rapidly stimulate intestinal Ca 2+ absorption thereby increasing the fraction of the dietary Ca 2+ that is absorbed by the intestine and made available to the skeletal system.
- these drugs effect the bone forming cells processes by stimulating bone formation which contributes to the amount of minerals present in bone.
- the analogs are formulated to achieve an oral dose equivalent to 0.5-25 micrograms of l ⁇ ,25 (OH) 2 D 3 /70 kg body weight, taken daily.
- the treatment duration is continuous for treatment of elderly patients and those with documented osteoporosis with serum Ca 2+ levels, urinary calcium excretion rates and alkaline phosphatase levels monitoring performed initially every two weeks and then on a monthly basis and bone mineral density determination at least once in every four months.
- Osteomalacia and rickets are caused by abnormal mineralization of bone and cartilage.
- Osteomalacia refers to the defect that occurs in bone in which the epiphyseal plates already have closed, therefore it is an adult disease
- rickets refers to the defect that occurs in growing bone, and it is therefore a disease of childhood.
- Abnormal mineralization in growing bone affects the transformation of cartilage into bone at the zone of provisional calcification. As a result, an enormous profusion of disorganized, nonmineralized, degenerating cartilage appears in this region, leading to widening of the epiphyseal plate and to swelling at the end of the long bones. Growth of the bone is retarded.
- Vitamin D-dependent rickets type I is a recessive disease in which there is a low level of 1,25 (OH) 2 D resulting from a selective deficiency in the renal production. To treat this condition, moderate doses of vitamin D (0.625 ⁇ g) or physiological doses (0.5-1 microgram) of l,25(OH) 2 D 3 are recommended.
- Vitamin D-dependent rickets type II is a hereditary condition in which there is a relatively high level of circulating 1,25 (OH) 2 D,however, due to a mutation in the vitamin D receptor which reduces the affinity of the receptor for its ligand l,25(OH) 2 D and therefore it does not function properly.
- large doses of l,25(OH) 2 D 3 (20-60 micrograms) are used.
- the serum Ca 2+ concentration in the disease state may be as low as 5.0-8.0 mg/100 ml.
- afflicted individuals typically have high levels of serum alkaline phosphatase, a marker for bone disease.
- serum alkaline phosphatase a marker for bone disease.
- any of the drug formulations of the l ⁇ ,25(OH) 2 D 3 conformationally flexible analogs which during testing were able to elicit both the rapid responses and genomic responses are suitable for treatment of osteomalacia.
- conformationally flexible analogs DE, DF, EV, GE, GF, HH, HJ, HL, HQ, HR, HS, IB, JR, JS, JV, JW, JX, JY and LO are effective drugs for treatment of osteomalacia.
- formulations comprising 6-s- ⁇ is locked analogs JM,
- the analog of the l ⁇ ,25(OH) 2 D 3 is formulated according to the conditions to be treated. Typically, the analog is administered orally or in a liquid form in an oral dose of equivalent to 0.25-2.0 micrograms dose of l ⁇ ,25(OH) 2 D 3 /70 kg body weight, daily. The dose is appropriately modified for children. The treatment duration depends on the treated conditions.
- Example 9 illustrates the treatment regimen.
- the child is treated with larger dosages of the analog and, its serum Ca 2+ levels are monitored weekly until the appropriate level is determined.
- the type II rickets can currently be treated only with gene therapy unless the analog of the invention is identified which is able to bind to the abnormal vitamin D receptor.
- Treatment of adult osteomalacia is achieved in the same manner as described for treatment of osteoporosis.
- Renal osteodystrophy is a bone disease that occurs in association with chronic renal failure.
- Chronic renal failure results from loss of the kidney ability to filter nitrogenous wastes from the blood for excretion in the urine.
- Chronic renal failure is a life threatening disease if the patient does not have regular access to hemodialysis.
- renal osteodystrophy develops because the normal endocrine function of the kidney is compromised resulting in an impairment of the 25(OH)D 3 -l- hydroxylase synthesis. This hydroxylase is responsible for the enzymatic production of the steroid hormone, l ⁇ ,25(OH) 2 D 3 .
- hormone D [l ⁇ ,25 (OH) 2 D 3 ] deficient.
- typical symptoms of hormone D deficiency namely impaired absorption of dietary calcium by the intestine occurs, leading to hypocalcemia and to increased secretion of parathyroid hormone (PTH) .
- PTH parathyroid hormone
- the PTH's secondary action in the instance of hypocalcemia is to stimulate the bone resorbing cells (osteoblasts) to mobilize bone calcium and make it available to the blood Ca 2+ pool.
- osteodystrophy The main components of renal osteodystrophy are osteitis fibrosa and osteomalacia.
- Osteitis fibrosa is a pathological condition which develops as a consequence of an increased level of parathyroid hormone and is characterized by an increase in bone resorption and marrow fibrosis. Renal osteodystrophy arises in part because of defective renal production of the active form of vitamin D in chronic renal failure, as discussed 5 above. Intestinal absorption of calcium is reduced. Low levels of l,25(OH) 2 D 3 in serum are observed.
- vitamin D metabolite are responsible for reduced absorption of calcium but they are also implicated in and directly affect the synthesis and secretion of parathyroid hormone by negating 0 the inhibitory effect of l,25(OH) 2 D 3 on a parathyroid hormone gene transcription.
- Treatment of these conditions is achieved by timely administration of the analog of the invention.
- any of the analogs belonging to the group of 5 conformationally flexible analogs or 6-s-cis locked analogs of l ⁇ ,25(OH) 2 D 3 are effective in stimulating the increase of intestinal Ca 2+ absorption and thus preventing a detrimental effect of parathyroid hormone leading to renal osteodystrophy.
- these analogs act on the osteoblast cells via 0 processes dependent upon both genomic events as well as rapid events to stimulate bone formation which contribute to the amount of bone mineral present and reverse the PTH stimulation of the osteoblasts.
- These analogs also act directly on the parathyroid gland to change the set-point relationship between 5 serum ionized Ca 2+ levels and the secretion of PTH.
- the parathyroid gland possess both VDR nuc and VDR mem which participate in the processes governing the secretion of PTH.
- the analog is formulated to achieve in oral dosage an equivalent of
- Psoriasis is a disorder of the skin characterized by dry, well-circumscribed silvery scaly papules and plaques of varying sizes. Psoriasis varies in severity from 1-2 lesions to a widespread dermatitis with disabling arthritis or exfoliation. Onset of psoriasis is usually between ages 10-40. While the general health of the individuals is not normally affected unless there is intractable exfoliation or severe widespread pustulation, psoriasis frequently creates in the afflicted individual a psychological stigma of an unsightly skin disease.
- Keratinocytes are the most important cells of the skin and they have been found to have both the nuclear [VDR nuc ] and membrane [VDRme m ] receptors for l ⁇ ,25 (OH) 2 D 3 . Under cell culture conditions, keratinocytes have been shown to display both genomic and rapid responses to l ⁇ ,25(OH) 2 D 3 and related analogs.
- the action of the vitamin D hormone (l ⁇ , 25 (OH) 2 D 3 ) and its analogs on keratinocytes growth and differentiation in psoriasis depends on an inappropriate stimulation of cell proliferation, on a decreased number of epidermal growth factor receptors, reduced levels of transforming growth factor ⁇ (TGF ⁇ ) , and abnormalities in the skin proteins keratin, involucrin and loricrin. These proteins are necessary for the formation of the cornified envelope, the normal structure of the upper skin layer. Psoriasis patient show a deficiency in production of these proteins. l ⁇ ,25(OH) 2 D 3 and its analogs have been shown in cell cultures of keratinocytes to stimulate the production of keratin, involucrin and loricrin.
- any of the formulations of the conformationally flexible analogs or 6-s-cis locked analogs which are active and stimulate the keratinocyte proliferation and production of keratin, involucrin or loricrin are effective in treating individuals with psoriasis.
- An analog is formulated for oral administration to achieve an oral dose equivalent to 0.5-2.0 micrograms of l ⁇ ,25(OH) 2 D 3 /70 kg body weight.
- the treatment is continuous, due to the continuous turnover and renewal of the keratinocytes of the skin.
- the suitability and efficacy of the treatment is monitored by following a progress of resolution of the external psoriatic plaques. Visual observations are often sufficient to evaluate the success of the treatment.
- a topical ointment, cream or solution (50 ⁇ g/gram) of the drug formulations of the l ⁇ ,25(OH) 2 D 3 conformationally flexible analogs or topical formulations of 6-s-cis locked analogs of l ⁇ ,25(OH) 2 D 3 , are used to treat individuals with external plaques of psoriasis.
- Leukemia is a rapidly progressing form of cancer of the white blood cells, which is characterized by replacement of normal bone marrow by blast cells of a clone arising from malignant transformation of a hemopoietic stem cell.
- the most responsive form of leukemia for treatment with l ⁇ ,25(OH) 2 D 3 analogs is acute myeloid leukemia (AML) .
- AML occurs at all ages and is the more common acute leukemia in adults. Diagnosis of AML is usually made via evaluation of the white cell types present in a blood sample.
- l ⁇ ,25(OH) 2 D 3 is known to be an effective inhibitor of human leukemia cell proliferation and as well a stimulator of the cell differentiation.
- Human leukemia NB4 cells have been shown to have both VDR- UC and DR mem and display both genomic and rapid responses to l ⁇ ,25(OH) 2 D 3 and its analogs.
- the drug formulation of the analog is oral or IV, containing 1-10 micrograms per day.
- the higher doses of the analog are administered intravenously or intraperitoneally. Treatment typically lasts
- the endpoints of the treatment are clinical biochemical determination of blood chemistries and particularly white blood cell morphology normalization. Because of their inhibitory action of human leukemia cell proliferation, analogs of the invention are especially effective in treating individuals with promyeloid leukemia.
- Prostate cancer is the most common non-skin cancer among men in many Western societies. Nearly 50% of all prostate cancers are advanced at the time of diagnosis and are incurable by surgery. Although many such cancers can be controlled by androgen withdrawal, there are no effective therapies for androgen-resistant disease. There is extensive objective evidence that l ⁇ ,25(OH) 2 D 3 induces prostate cancer cells to experience an inhibition of proliferation as well a selective differentiation. A variety of animal models of prostate cancer have been studied and are available as seen in Table 7.
- Prostate cells are known to possess the VDR nuc and VDR mem .
- the analogs of the invention are effective in treating individuals with prostate cancer.
- the dose regimen depends on the advanced state of the cancer. Doses are higher than renal osteodystrophy, typically 5-10 ⁇ g daily or more. The drug is administered either IV, IP or orally 3X weekly for several months. A major endpoint is a measurement of the presence of the prostate antigen in serum, which will be reduced if the drug is effective.
- Analogs Utility For Organ Transplantation The vitamin D endocrine system includes the immune system in its sphere of actions. Both activated T and B lymphocytes have the VDR nuc and VDR m ⁇ m .
- vitamin D-deficient animals and humans have a higher risk of infection, related to deficient macrophage function, whereas the monocytes/macrophage differentiation (tumor cell cytotoxicity, phagocytosis, mycobactericidal activity) is enhanced by l ⁇ , 25 (OH) 2 D 3 .
- the natural killer cell activity is also enhanced by l ⁇ ,25(OH) 2 D 3 .
- This enhancing effect of the nonspecific immune defense contrasts with an inhibition of the antigen-specific immune system as demonstrated by a decreased T cell proliferation and activity.
- the antigen production by B cells can also be decreased by treatment with l ⁇ ,25(OH) 2 D 3 .
- Table 7 several animal models have been used to evaluate the effect of l ⁇ ,25(OH) 2 D 3 and its analogs on organ transplantation and rejection. These results support utilizing analogs of l ⁇ ,25(OH) 2 D 3 to counter immunoreactions connected with human organ transplantation, such as kidney transplantation, heart, or combined heart and lung transplantation, skin transplantation, and pancreas transplantation.
- the analog HL [l ⁇ ,25(OH) 2 D 3 ] which is an antagonist for the rapid actions mediated by l ⁇ ,25(OH) 2 D 3 is suitable to treat individuals experiencing hypercalcemia, particularly individuals with elevated plasma levels of l ⁇ ,25(OH) 2 D 3 occurring in primary hyperparathyroidism or drug overdose of l ⁇ ,25(OH) 2 D 3 or l ⁇ ,(OH)D 3 with drugs Rocaltrol or Alpherol.
- the clinical hypercalcemia describes circumstances where the blood concentration of Ca 2+ is elevated above the normal range of 9.0-10.5 mg Ca 2+ /100 serum. Elevations of blood Ca 2+ concentration above 12.0-13.0 mg/100 ml is cause for grave concern, and if left untreated it becomes life threatening as it can lead to tachycardia. Individuals who are found to have serum Ca 2+ levels above 12.0-13.0 mg/100 ml are frequently treated by hemodialysis with a low concentration of Ca 2+ in the dialysis bath in an effort to acutely lower their prevailing serum concentration of Ca 2+ to the normal range.
- Ca 2+ mobilizing activity This process results in additional Ca 2+ being made available to the blood compartment from both the intestine dietary Ca 2+ and bone calcium (hydroxyapatite mineral) , which is likely to result in hypercalcemia.
- the present invention also relates to pharmaceutical compositions useful for treating vitamin D disorders.
- These compositions comprise an effective amount of the analog of the invention or the pharmaceutically acceptable salt thereof in acceptable, non-toxic carriers.
- composition may comprise solely of the one analog or an admixture of two or more analogs of the invention or a pharmaceutically acceptable salt thereof in a suitable amount to treat a subject and/or condition.
- composition may include any suitable conventional pharmaceutical carrier or excipient as well as other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.
- Activity of vitamin D and its metabolites is typically expressed as one international unit.
- One international unit corresponds to 1/40 of a microgram, that is 40 international units are equal to 1 microgram or 65 pmoles of vitamin D.
- the amount of the analog in the composition will depend on its relative activity vis-a-vis to the activity of vitamin D and particularly to its metabolite l ⁇ ,25 (OH) 2 D 3 .
- the analogs of the invention may be formulated with or in suitable pharmaceutical vehicles known in the art to form particularly effective pharmaceutical composition. Generally, an effective amount of active analog is about 0.001%/w to about 510%/w of the total formulated composition. The rest of the formulated composition will be about 90%/w to about 99.999%/w of a suitable excipient.
- compositions of the analog of the invention particularly suitable for oral administration conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium 5 carbonate, and the like may be used.
- a pharmaceutically acceptable nontoxic composition is formed by the incorporation of any of the normally employed excipients, such as those named above.
- Such oral compositions take the form of solids, solutions or 0 suspensions, such as tablets, pills, capsules, powders, sustained release formulations and the like.
- Such compositions may contain 0.1%-95% of active ingredient, preferably l%-70%.
- binders and carriers include for 5 example polyalkylene glycols or triglycerides .
- Such suppositories may be formed from mixtures containing active ingredient in the range of 0.5%-10%, preferably 1-2%.
- Liquid pharmaceutically administrable compositions suitable for oral or parenteral administration can, for example, be 0 prepared by dissolving, dispersing, suspending, etc., the analog in a suitable carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
- a suitable carrier such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
- the carrier may optionally contain pharmaceutical adjuvants.
- the 5 pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
- Parenteral compositions are typically liquid compositions suitable for subcutaneous, intraperitoneal, intramuscular or intravenous administration.
- Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
- Suitable excipients are, for example, water, saline, destrose, glycerol, ethanol or the like.
- compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
- compositions and dosage forms are known, or will be apparent, to those skilled in this art.
- compositions of the invention see Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pennsylvania, 15 th Edition, 1975.
- the composition or formulation to be administered will, in any event, contain a quantity of the analog (s) in an amount effective to alleviate the disease symptoms of the subject being treated.
- the invention also relates to a mode of administration of the compounds of the invention.
- compositions described hereinafter can be via any of the accepted modes of administration for such agents suitable for treatment of diseases which affect the vitamin D endocrine system. These methods include oral, parenteral and other systemic administration.
- the composition may be in the form of solid, semi-solid or liquid dosage forms, such as, for example. tablets, suppositories, pills, capsules, powders, liquids, suspension, drops or the like, preferably in unit dosage forms suitable for single administration of precise dosages.
- Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Parenteral administration also includes the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained.
- the amount of active compound administered depends on the subject being treated, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. However, an effective dosage will be in the range of 0.001-15 ⁇ g/kg/day, preferably 0.01-3 ⁇ g/kg/day. For an average 70 kg human, this would amount to 0.07-1000 ⁇ g per day, or preferably 0.7-210 ⁇ g/day.
- the analogs of the invention are potent agonist for the genomic responses or antagonists of the rapid nongenomic responses connected with the biological action of vitamin D 3 . They are therefore useful for treatment and prevention of diseases connected with either insufficiency or with overproduction of 1 ⁇ ,25- dihydroxy vitamin D 3 .
- Scheme A shows synthesis of analogs DE, DF and EV described in Examples 1-3.
- the A-ring fragment 1 (0.077 g, 0.14 mmol) and 0.060 g (0.16 mmol) of the CD ring triflate (2a) were dissolved in 0.6 L of dry DMF.
- Bis-triphenylphosphine palladium dichloride complex (Pd(PPh 3 ) Cl 2 , 3 mg) and diethyla ine (0.076 mL, 0.55 mmol) were then introduced.
- the mixture was heated to 80°C 5 for 5 h and then after cooling to room temperature, water was added and the mixture was extracted with ether.
- the combined ether extracts were washed successively with a solution 10% HCl, a solution saturated of NaHC0 3 and brine. After drying (MgS0 4 ) and concentrating the solution, the crude residue was
- the CD-ring triflate 2b (0.053 g, 0.1 mmol) and the A- ring 1 (0.046 g, 0.12 mmol) were dissolved under argon in 0.4 mL of dry DMF (distilled from benzene and then from BaO) .
- Diethylamine (0.054 mL, 0.39 mmol) and bistriphenylphosphine palladium dichloride (2 mmol, 2 mg, Pd (PPh 3 ) 2 C1 2 ) were added 5 and the mixture was heated at 80°C for 4.5 h.
- the solution was cooled and then diluted with ether.
- the organic layer was separated, washed with a solution 10% HC1, a saturated solution of NaHC0 3 and then brine. After drying (MgS0 4 ) and concentrating, the residue was purified by HPLC (Rainin
- HEMS m/z 762.5289 (calcd. for C 46 H 78 0 3 Si 3 , 762.5259).
- CD ring triflate 2c (0.032 g, 0.06 mmol) and A-ring enyne 1 (0.025 g, 0.06 mmol) were stirred in DMF (0.4 mL) in the presence of 1.5 mg of Pd(PPh 3 ) 2 (OAc) 2 , 1 mg of cuprous iodide and 0.4 L of Et 2 NH. After stirring the mixture for 2 h at room temperature, water was added and the mixture was extracted with ether. The combined ether extracts were washed with a 10% solution of HC1, a saturated solution of NaHC0 3 and brine.
- HEMS 762.5207 (calcd. for C 46 H 78 0 3 Si 3 , 762.5259).
- Dienyne 3c (0.020 g, 0.026 mmol) was dissolved in 13 mL of EtOAc and 42 ⁇ L of a solution of quinoline (0.17 M in hexanes) and then 42 mg of Lindlar catalyst were added. The mixture was stirred for 1 h under a positive pressure of
- the reaction mixture was stirred at room temperature for 1.5 h under argon. Ether was added and the mixture was washed with H 2 0 (3 x 5 mL) , dried (MgS0 4 ) and evaporated in vacuo.
- the crude dark brown oil was purified by flash chro atography (10% ethyl acetate-hexane) to afford after vacuum drying 102 mg (79%) of the dienyne 3 as a viscous oil, which was sufficiently pure for the next step.
- HEMS m/z 416.3288 (calcd. for C 27 H 44 0 3 , 416.3292).
- MS m/z 416 (21, M) , 398 (72, M - H 2 0) , 380 (36, M - 2H 2 0) , 362 (3), 329 (3), 285 (11), 251 (10), 227 (9), 197 (8), 152 (29, A-ring portion after C 7/8 -cleavage) , 134 (base, m/z 10 152 - H 2 0) .
- Analog HO Chemical Synthesis of Analog HO
- analog HQ namely (22S) -l ⁇ , 25-Dihydroxy-22 ,23,23, 24-tetradehydrovitamin D 3
- Analog HO is prepared according to Scheme D.
- Bi ⁇ (triphenylphosphine) palladium(II) acetate (5.0 mg, 6.7 mmol) and copper (I) iodide (4.8 mg, 25.2 mmol) were added at ambient temperature to a mixture of enol triflate 2a (54.8 mg, 0.105 mmol), enyne 1 (48.0 mg, 0.126 mmol) in DMF (1.0 mL) and diethylamine (1.0 mL) under an argon atmosphere. The mixture was stirred at room temperature for 2.5 h after which time ether (10 mL) was added and the mixture washed with brine (3 x 10 mL) .
- the organic layer was dried (MgS0 4 ) , filtered and concentrated to afford a dark brown residue.
- the crude product was passed down a short silica gel column (15% ethyl acetate/hexanes) followed by HPLC separation (Rainin Dynamax, 1.0 x 25 cm, 8 ⁇ m, 1% ethyl acetate/hexanes) to afford after drying, spectro ⁇ copically homogeneou ⁇ dienyne 3a (59 mg, 75%) a ⁇ a colorless oil.
- X H___ME ⁇ _0.06 (6H, Si-Me 2 , s) , 0.07 (6H, Si-Me 2 , s) , 0.09 (6H, Si-Me 2 ) , 0.72 (3H, C 18 -Me, s) , 0.85 (9H, t-Bu, s) , 0.88 (9H, t-Bu, s) , 0.89 (9H, t-Bu, s) , 1.09 (3H, C 21 -Me, d, J-6.6 Hz), 1.30 (3H, C 26,27 -CH 3 , s) , 1.31 (3H, s) , 1.90 (3H, C 19 -Me, br s) , 4.09 (IH, H 3 , broad m, W ⁇ 15 Hz) , 4.19 (IH, H l m) , 5.18 (IH, H 22 , dd, J ⁇ 6.6 Hz, 6.6 Hz) , 5.28 (
- 1 H _ ⁇ ME: ⁇ _0.05 (3H, Si-Me, s) , 0.06 (3H, Si-Me, s) , 0.07 (6H, Si-Me 2 , s) , 0.09 (6H, Si-Me 2 , s) , 0.71 (3H, C 18 -Me, s) , 0.85 (9H, t-Bu, s) , 0.886 (9H, t-Bu, ⁇ ) , 0.895 (9H, t-Bu, s) , 1.09 (3H, C 21 -Me, d, J ⁇ 6.6 Hz), 1.30 (3H, C 26,27 -Me, s) , 1.31 (3H, C 26/27 -Me, s) , 1.65 (IH, C 19 -Me, br ⁇ ) , 4.01-4.10 (IH, H 3 , m), 4.11 (IH, Hi, br s) , 5.17 (IH, H 22 ,
- 1 H MME: ⁇ _0.57 (3H, C 18 -Me, s) , 1.08 (3H, C 21 -Me, d, J ⁇ 6.6 Hz), 1.34 (6H, C 26(27 -2CH 3 , s) , 2.32 (IH, H 4 ⁇ , dd, J " 13.2 Hz, 6.0 Hz), 2.60 (IH, H 4 ⁇ , dd, J ⁇ 13.2 Hz, 3.0 Hz), 2.83 (IH, H 9(3 , dd, J ⁇ 11.7 Hz, 3.0 Hz), 4.23 (IH, H 3 , m, " 20 Hz), 4.43 (IH, Hi, m, " 12 Hz), 5.00 (IH, H 19z , narrow m) , 5.33 (IH, H 19E , narrow m) , 5.28-5.35 (2H, H 22 and H 24 , m, partially obscured by H 19E ) , 6.02 and 6.38 (2H, H
- previtamin 4b 15 mg, 19.9 mmol
- isooctane 10 mL
- the solvent was removed under vacuum to give a colorless residue, which after HPLC separation (Rainin Dynamax, 0.1% ethyl acetate/hexanes) afforded a 9:1 mixture of vitamin and previtamin.
- the mixture was dissolved in THF (1 mL) and treated with tetrabutylammonium fluoride (273 ⁇ L, 1.0 M in THF, 0.273 mmol) at room temperature for 15 h, protected from the light.
- Scheme E relates to the analog HS described in Example 11.
- a solution of A-ring phosphine oxide 1 (122 mg, 0.21 mmol) 5 in dry THF (3 mL) was treated with n-butyllithium (0.14 mL, 0.21 mmol, 1.55 M in hexanes) and then with CD-ring ketone 2 (57 mg, 0.14 mmol) in dry THF (2.2 mL) . After work up, there was obtained 81 mg (83%) of the protected vitamin 3 of sufficient purity for the next step.
- the analytical data for the precursor is:
- the analog HS (5) was prepared by adding tetra-n-butyl- ammonium fluoride (2.16 ⁇ L, 0.216 mmol, 1 M in THF) to a solution of the protected alcohol precursor compound 4 (18.1 mg, 0.024 mmol) in anhydrous THF (2 mL) . The mixture was stirred for 20 hours at room temperature, then concentrated to dryness. The resulting crude material was directly flash chromatographed through a short column of silica gel (EtOAc) and then purified by HPLC (Rainin Dynamax, 1.0 x 25 cm, 8 ⁇ m ⁇ ilica column, EtOAc) to give, after vacuum drying, the analog 5 HS (5, 7 mg, 70%) as a white foam.
- EtOAc silica gel
- the analytical data for the analog HS (5) is: 1 H__MB (300 MHZ): (CD 3 OD) ⁇ 1.07 (3H, C 21 -CH 3 , d, J _ 6.4 Hz), 1.16 (6H, C 26 , 27 -CH 3 , s) , 1.0-2.2 (remaining ring and side chain hydrogens, series of m) , 2.24 (IH, dd, J " 13.2 Hz, 7.2 10 Hz) , 2.51 (2H, br d, J " 13.0 Hz), 2.91 (IH, br d, J “ 11.2 Hz), 3.35 (2H, H 18 , d, J " 11.8 Hz), 3.41 (IH, H 18 , d, J ⁇ 11.8 Hz), 4.10 (IH, H 3 , m) , 4.34 (IH, H x , t, J ⁇ 5.6 Hz), 4.87 (IH, Hig, s) , 5.28 (IH
- HEMS m/z 432.3242 (calcd. for C 27 H 44 0 4 , 432.3241).
- Scheme F relates to the analog IB described in Example 12.
- EXAMPLE 17 ⁇ Ch_TTicq i Syn h sis of Analog TT.
- This example illustrates preparation of the analog IB, namely 23 - ( m- d imethylhydroxymethyl) phenyl) -22-yne- 2 ,25, 26 , 27 - tetranor- l ⁇ ( OH)D 3 , as seen in Scheme F.
- step l i and 2 are reacte d in the presence o f pa l la d i ⁇ rm (O) resulting in 3, which was obtaine d pure b y f las h chromatography using the solvent 20% ethyl acetate in hexane.
- step 2 55 g of the product of step 1 was reacte d with 183 mg pyridinium chlorochro ate (PDC) , 1 2 mg pyri d iniu trifluoroacetate (PT FA) and 100 L C H 2C 1 2 accor d ing to a standard procedure.
- PDC pyridinium chlorochro ate
- PT FA pyri d iniu trifluoroacetate
- Scheme H relates to analogs JR, JS, JV and JW described in Examples 17-20. 35 Scheme H
- IZY (100% EtOH) ⁇ _ ax 266 nm (e 15,000); ⁇ - in 228 nm (e 9,300).
- HEMS m/z 416.3281 (calcd. for C 27 H 44 0 3 ,- 416.3292).
- MS m/z 416 (8), 398 (10), 380 (17), 362 (8), 347 (6), 306 (2), 267 (7), 251 (41), 225 (10), 197 (30), 181 (11), 131 (25), 105 (57), 91 (49), 81 (32), 69 (56), 59 (base).
- 1 H____E (300 MHZ): (CDC1 3 ) ⁇ 0.06 (6H, Si-2CH 3 , s) , 0.09 (6H, Si-2CH 3 , s) , 0.10 (9H, Si-3CH 3 , s) , 0.9-1.0 (24H, series of overlapping signals due to 2 Si-tBu, C 18 -CH 3 and C 2 ⁇ CH 3 ) , 1.20 (6H, C 26/27 -CH 3 , S) , 1.87 (3H, C 19 -CH 3 , br s) , 0.97-2.39 25 (remaining ring and side chain hydrogens, ⁇ eries of m) , 4.03- 4.12 (IH, Ci-H, m, VT26.7 Hz), 4.17 (IH, C 3 -H, br s) .
- the organic layer was washed with NaHC0 3 (2 x 5 L) and brine (1 x 5 mL) and dried (MgS0 4 ) .
- the concentrated oil was purified by flash chromatography (silica gel, 2.5% ethyl acetate/hexanes) followed by HPLC (2.5% ethyl acetate/hexanes, Rainin Dynamax column, 8 mL/min flow rate) to afford pure benzoate 3 (405 mg, 59%) and recovered propargyl alcohol (156 mg, 27%) , in that order of elution.
- the propargyl benzoate 3 was characterized by spectroscopic analysis.
- HEMS (FAB) m/z 835.5564 (calcd. for C 49 H 84 0 5 Si 3 minus H, 835.5551) .
- MS m/z 836 (2), 716 (13), 675 (2), 584 (12), 541 (2), 493 (4), 463 (4), 437 (5), 355 (8), 301 (9), 223 (11), 179 (30), 131 (59), 105 (base).
- the product was purified by flash chromatography (silica gel, 2% ethyl acetate/hexanes) followed by HPLC (2% ethyl acetate/hexanes, Rainin Dynamax column, 8 mL/min flow rate) to afford silyl protected vinylallene (1S,3R, 6S) -1, 3-di(tert-butyldimethylsilyloxy) -25- trimethylsilyloxy-9, 10-secocholesta-5 (10) ,6,7-triene (0.3085 g, 75.5%) .
- the product was identified only by ' ⁇ -NMR analysis and immediately deprotected as described below. This material appeared to be more stable as the triol 4.
- HEMS m/z 416.3277 (calcd. for C 27 H 44 0 3 , 416.3292).
- MS m/z 416 (10), 398 (10), 380 (9), 365 (4), 342 (2), 328 (2), 313 (2), 287 (5), 269 (5), 251 (8), 197 (7), 159 (15), 134 (54), 105 (32), 95 (29), 81 (38), 69 (40), 59 (base) .
- HY_ (100% EtOH) ⁇ na - 242 nm (e 22,300), 234 nm (e 22,100) .
- HEMS m/z 416.3291 (calcd. for C 27 H 44 0 3 , 416.3292).
- MS m/z 416 (25, M) , 398 (20), 380 (26), 365 (7), 347 (5), 325 (5), 313 (3), 287 (11), 269 (13), 251 (38), 225 (12), 213 (14), 197 (26), 173 (19), 159 (25), 145 (32), 133 (35), 105 (47), 95 (33), 81 (38), 69 (47), 59 (base).
- Scheme I relates to analogs JX and JY de ⁇ cribed in Examples 21 and 22.
- the A-ring pho ⁇ phine oxide 1, (48 mg, 0.11 mmol) in dry THF (1.8 mL) was cooled to -78°C and n-butyllithium (1.5 M in hexanes, 0.074 mL, 0.11 mmol) wa ⁇ added dropwi ⁇ e via a syringe.
- the resulting deep red solution was stirred for 10 min and then treated with a solution of CD-ring ketone 2a (28 mg, 0.070 mmol) in dry THF (0.6 mL) via cannula.
- the mixture was ⁇ tirred 2 h at -78°C, warmed to room temperature and quenched with water (5 L) .
- the aqueous layer was ⁇ eparated and extracted with EtOAc (3 x 5 L) .
- the combined organic layers were washed with brine, dried over Na 2 S0 4 , and concentrated.
- the crude residue was purified by rapid filtration through a short ⁇ ilica gel column (20% EtOAc/hexanes) to afford 20.1 mg (46%) of the protected vitamin 3a.
- the latter (20.1 mg, 0.0315 mmol) in THF (1 mL) was placed under argon and TBAF (0.32 mL, 1 M in THF, 0.32 mmol) was added dropwise. After stirring for 18 h, the solvent was partially evaporated and the residue diluted with water (5 mL) .
- the solution was stirred 10 min and then treated dropwise with a solution of CD-ring ketone 2b (41 mg, 0.102 mmol) in dry THF (0.85 mL) .
- the mixture was stirred 2 15 h at -78°C and then allowed to warm to room temperature over 1 h.
- the solvent was partially evaporated and then quenched with 5 mL water.
- the aqueous layer was separated and extracted with EtOAc (3 x 5 mL) .
- the combined organic layers were washed with brine, dried over Na 2 S0 4 and concentrated.
- Lithium di-isopropyl amide (LDA) was prepared by the addition of di-isopropyl amine (0.097, 0.69 mmol) to a ⁇ olution of n-BuLi in hexane ⁇ (0.48 mL, 1.6 M, 0.77 mmol) and 15 dry THF (1 mL) at -78°C. After ⁇ tirring for 10 min at -78°C and at room temperature for 15 min the ⁇ olution was again cooled to -78°C and the 25-TMS cyclopropylketone 4 (0.200 g, 0.548 mmol) in THF (2 mL) was added dropwise via a cannula.
- LDA Lithium di-isopropyl amide
- 1 H___MB (300 MHZ, CDC1 3 ) : ⁇ 0.06 (s, 6H, SiMe 2 ) , 0.09 (s, 20 6H, SiMe 2 ) , 0.10 (s, 9H, SiMe 3 ) , 0.46 (dd, J ⁇ 7.5,-4.0 Hz, IH, H a ) , 0.59 (apparent t, J ⁇ 3.6 Hz, IH, H b ) , 0.88 (s, 9H, Si fc Bu) , 0.89 (s, 9H, Si' ⁇ u) , 0.90 (superimposed signal, 3H, C 21 -Me) , 0.92 (s, 3H, C ⁇ 8 -Me) , 0.94-2.43 (remaining ring and side chain hydrogens, series of m) , 1.19 (s, 6H, C 26(27 -2Me) , 1.86 (br s, 25 3H, C 19 -Me) , 4.08
- NBA+Na, 37 25 329 (NBA+Na, 37), 307 (NBA, 23), 289 (NBA, 14), 192 (NBA+K, 39), 176 (NBA+Na, base), 154 (NBA, 86), and 136 (NBA, 61).
- This example describes a ligand receptor competitive assay used for determination of an analog's relative ability to bind to VDR nuc expressed as relative competitive index (RCI) .
- RCI relative competitive index
- the relative affinity of nonradioactive l ⁇ ,25(OH) 2 D 3 and each analog to compete with [ 3 H]l ⁇ ,25(OH) 2 D 3 for binding to the VDR nuc of NB4 cells was carried out in vitro .
- the NB4 cells were collected from a fast growing ⁇ tage and the cellular VDR nuc of l ⁇ ,25(OH) 2 D 3 were extracted from KTED buffer containing 10 mM Tris-HCI, pH 7.4, 300 mM KC1, ImM EDTA and 5 mM DTT. After sonication, the cell extract was further centrifuged at 500 x g for 10 min. The supernatant was collected for use in a ligand-receptor binding assay.
- Relative Competitive index This example describes a Relative Competitive Index Assay used for determination of analogs binding affinity to vitamin D-binding protein. Binding of the l,25(OH) 2 D 3 and its analogs to the human vitamin D-binding protein (hDBP) was performed at 4°C essentially as described previously in the Journal of Biological Chemistry 267; 3044-3051 (1992).
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Abstract
Description
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Priority Applications (4)
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AU95035/98A AU9503598A (en) | 1997-09-26 | 1998-09-23 | Therapeutically effective 1alpha, 25-dihydroxyvitamin D3 analogs |
JP2000513586A JP2001517706A (en) | 1997-09-26 | 1998-09-23 | Therapeutically effective 1α, 25-dihydroxyvitamin D3 analogs |
EP98948468A EP1021193A1 (en) | 1997-09-26 | 1998-09-23 | THERAPEUTICALLY EFFECTIVE 1$g(a), 25-DIHYDROXYVITAMIN D 3? ANALOGS |
CA002304056A CA2304056A1 (en) | 1997-09-26 | 1998-09-23 | Therapeutically effective 1.alpha., 25-dihydroxyvitamin d3 analogs |
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US60/060,173 | 1997-09-26 | ||
US09/073,723 US6103709A (en) | 1993-12-23 | 1998-05-07 | Therapeutically effective 1α,25-dihydroxyvitamin D3 analogs and methods for treatment of vitamin D diseases |
US09/073,723 | 1998-05-07 | ||
US09/074,565 US6121469A (en) | 1993-12-23 | 1998-05-07 | Therapeutically effective 1α,25-dihydroxyvitamin D3 analogs |
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PCT/US1998/019862 WO1999016452A1 (en) | 1997-09-26 | 1998-09-23 | THERAPEUTICALLY EFFECTIVE 1α, 25-DIHYDROXYVITAMIN D3 ANALOGS |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1021193A1 (en) |
JP (1) | JP2001517706A (en) |
AU (1) | AU9503598A (en) |
CA (1) | CA2304056A1 (en) |
WO (1) | WO1999016452A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10406202B2 (en) | 2014-10-22 | 2019-09-10 | Extend Biosciences, Inc. | Therapeutic vitamin D conjugates |
US10420819B2 (en) | 2014-10-22 | 2019-09-24 | Extend Biosciences, Inc. | Insulin vitamin D conjugates |
CN115850142A (en) * | 2022-11-25 | 2023-03-28 | 甘肃皓天医药科技有限责任公司 | Synthesis method of 7Z vitamin D compound |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5395829A (en) * | 1990-10-04 | 1995-03-07 | Trustees Of Boston University | Compositions comprising vitamin D lumisterol analog precursors |
-
1998
- 1998-09-23 WO PCT/US1998/019862 patent/WO1999016452A1/en not_active Application Discontinuation
- 1998-09-23 CA CA002304056A patent/CA2304056A1/en not_active Abandoned
- 1998-09-23 EP EP98948468A patent/EP1021193A1/en not_active Withdrawn
- 1998-09-23 AU AU95035/98A patent/AU9503598A/en not_active Abandoned
- 1998-09-23 JP JP2000513586A patent/JP2001517706A/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5395829A (en) * | 1990-10-04 | 1995-03-07 | Trustees Of Boston University | Compositions comprising vitamin D lumisterol analog precursors |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10406202B2 (en) | 2014-10-22 | 2019-09-10 | Extend Biosciences, Inc. | Therapeutic vitamin D conjugates |
US10420819B2 (en) | 2014-10-22 | 2019-09-24 | Extend Biosciences, Inc. | Insulin vitamin D conjugates |
US10702574B2 (en) | 2014-10-22 | 2020-07-07 | Extend Biosciences, Inc. | Therapeutic vitamin D conjugates |
US11116816B2 (en) | 2014-10-22 | 2021-09-14 | Extend Biosciences, Inc. | Therapeutic vitamin d conjugates |
CN115850142A (en) * | 2022-11-25 | 2023-03-28 | 甘肃皓天医药科技有限责任公司 | Synthesis method of 7Z vitamin D compound |
Also Published As
Publication number | Publication date |
---|---|
EP1021193A1 (en) | 2000-07-26 |
CA2304056A1 (en) | 1999-04-08 |
AU9503598A (en) | 1999-04-23 |
JP2001517706A (en) | 2001-10-09 |
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