CA2304056A1 - Therapeutically effective 1.alpha., 25-dihydroxyvitamin d3 analogs - Google Patents

Abstract

Novel analogs of 1.alpha., 25-dihydroxyvitamin D3, which are selective agonists for the genomic responses or agonists or antagonists for the rapid cellular responses in a wide array of diseases in which 1.alpha., 25-dihydroxyvitamin D3 or its prodrugs are involved. Novel analogs have general formulae represented by compounds of groups I-V. A method for treatment and prevention of diseases connected with the endocrine system.

Description

THERA_pECT'I'ICALT_.y EFFECTIVE
i a,,. - -D HyD O~X ,VTTAMTN D3 ANALOGS
EACKGROUND OF THE INVEN TON
The current invention concerns novel analogs of 1a,25 dihydraxyvitamin D3 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 1a,25-dihydroxyvitamin D3 or its prodrugs are involved. In particular, 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 D3 metabolites. In particular, the current invention concerns therapeutic properties of 1a,25-dihydroxyvitamin D3 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,~hosphorus.
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, 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; and for treatment of any other disease in which 1a,25-dihydroxyvitamin D3 or its pro-drugs are involved.

The deficiency or overproduction of vitamin D3 metabolites result in serious disturbance of homeostasis by vitamin D endocrine system.
Analogs of vitamin D, metabolites act rapidly, specifically, and in the same manner as the vitamin D3 metabolites on the genomic cellular apparatus and also elicit rapid nongenomic responses correcting the vitamin D, caused deficiencies.
The certain analogs of 1a,25(OH)2D3 have biological activities similar to those of 1a,25(OH)ZD, 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 1a,25(OH)Z-7-dehydrocholesterol; analog JN, namely, 1a,25(OH)2-lumisterol3; analog JO, namely, 1a,25(OH)2-pyrocalciferol3; analog JP, namely, 1a,25(OH)2-isopyrocalciferol3; analog HS, namely, la, 18, 25 (OH) 3-D3;
analog GE, namely, 14-epi-1, 25 (OH)Z-D3; analog-GF; namely, 14-epi-1,25(OH)2-pre-D3; analog JR, namely, 1 a,25{OH)2-7,8-cis-D3; analog JS, namely, 1,25(OH)Z-5,6-traps-7,8-cis-D3;
analog HH, namely, 1(3,25(OH)2-Epi-D3; analog HJ, namely, 1a,25(OH)2-3-Epi-D3; analog JV, namely, (iS,3R,6S)-7,19-retro-1, 25 (OH) 2-D3 or (6(3) -1, 25-vinylallene) ; analog JW, namely, (1S,3R,6R)-7,19-retro-1,25(OH)2-D3, or [(6a)-1,25-vinylallene]; analog JX, namely, 22-(p-hydroxyphenyl)-23,24,25,26,27-pentanor-D3; analog JY, namely, 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor-D3; analog IB namely 23-[m(dimethylhydroxyethyl)phenyl]-22-yne-24,25,26,27-tetranor-la-hydroxy-D3, analog LO, namely l4a,l5a-methano-la, 25 (OH) 2D3.
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 raith or caused by vitamin D3 deficiency or overproduction, by providing a subject in need of such treatment a vitamin D3 analog which is either an agonist of vitamin D3 or its antagonist, wherein the analog is selected from the group of compounds 5 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 1a,25(OH)ZD3 which is present in target l0 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 15 organs during homeostatic responses and correction of pathological conditions in Which the vitamin D3 or 1a,25(OH)ZD3 are involved by analogs of the invention.
Another aspect of this current invention is the rapid nongenomic stimulation of mitogen-activated protein kinase 20 (MAP-kinase) in chick intestinal and human leukemic cells.
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.
25 Still another aspect of the invention is a method for treatment of diseases caused by deficiencies or overproduction of 1a,25(OH)2D3 or treatment of its functional deficiencies by providing a subject in need of correcting these deficiencies with an agonist analog of the 30 1a,25(OH)zD3 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.
35 Another aspect of the present invention involves controlling the rapid nongenomic responses mediated by 1a,25(OH)2D3 by treating the subject in need of such treatment with an antagonist analog which is i~i,25(OH)2D3.
Another aspect of the current invention is 1a~25-dihydroxyvitamin D3 and its 6-s-cis analogs which are selective agonists for the activation of MAP-kinase.
5 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 10 diseases, such as Alzheimer's; parathyroid diseases, such a s hyperparathyroidism, hypoparathyroidism, pseudoparathyroidism or secondary parathyroidism; liver and pancreas diseases, such as diabetes, cirrhosis, obstructive jaundice or drug-induced metabolism; intestine diseases, 15 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 20 sarcoidosis; or any other disease - in which 1a,25-dihydroxyvitamin D3 or its pro-drugs are involved.
Another aspect of the current invention is a method for treatment of vitamin D3 deficiencies by providing 1a,25-dihydroxyvitamin D3 analogs which are selective 25 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 30 pharmaceutical composition comprising a 1a,25-dihydroxyvitamin D3 analog useful for treatment of rickets, osteomalacia, osteoporosis, osteopenia, osteosclerosis, renal osteodystrophy, psoriasis, medullary carcinoma, Alzheimer's, hyperparathyroidism, 35 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.
5 Another aspect of the current invention is a method for treatment of the above-listed diseases wherein the analog is selected from the group consisting of analog JM, namely ia,25(OH)27-dehydrocholesterol; analog JN, namely, 1a,25(OH)2-lumisterol3; analog JO, namely, 10 1a,25(OH)2-pyrocalciferol3; analog JP, namely, 1a,25(OH)2-isopyrocalciferol3; analog HS, namely, la,18, 25 (OH) 3-D~; analog GE, namely, 14-epi-1, 25 (OH) 2-D3;
analog GF, namely, 14-epi-1,25(OH)2-pre-D3; analog JR, namely, 1a,25(OH)Z-7,8-cis-D3; analog JS, namely, 15 1a,25(OH)2-5,6-traps-7,8-cis-D3; analog HL, namely, 1(3, 25 (OH) Z-D3; analog HH, namely, 1a, 25 (OH)2-3-epi-Dj ; analog HJ, namely, 1a,25(OH)2-epi-D3; analog JV, namely, (iS,3R,6S)-7,19-retro-1,25(OH)Z-D ~, or (6-((3)-1,25-vinylallene; analog JW, namely, 20 ( 1S, 3R, 6S) -7, 19-retro-1, 25 (OH) Z--~ 3 or (6-(a)-1,25-vinylallene; analog JX, namely, 22-(p-hydroxyphenyl)-23,24,25,26,27-pentanor-D3; analog JY, namely, 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor-D3; and analog IB, namely 23-[m(dimethylhydroxymethyl) 25 phenyl]-22-yne-24, 25, 26, 27-tetranor-la-hydroxy-D3.
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 30 organs during homeostatic responses and correction of pathoivgical conditions in which the vitamin D3 or 1a,25(oH)ZD3 are involved by providing a subject in need thereof an analog of the invention.
Still yet another aspect of the current invention is a 35 pharmaceutical composition comprising an analog of the invention or its pharmaceutically acceptable salt.

As defined here:
"Steroid-like conformation", the seco-B ring can assume, in the limit, one of two conformations as a 5 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-traps orientation (D), 10 the A ring is present in an "extended conformation."
"Alpha", or "a", "beta" or "~3" position or configuration mean the absolute configuration notation used in steroids, such as cholesterol or in natural products;
the term "a" or "~i" mean the carbon or the substituent, as 15 the case may be, within the context of the structural formulas presented herein.
"Cis" or "traps" terms are used in reference to vitamin D3 which is 5,6-cis/7,8-traps. Terms "Z" or "E"
designations are less desirable because these designations 20 are reversed when a C1 hydroxyl is present. -"6-traps 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.
25 "Agonist" 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.
30 "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 35 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.
"Agonist" 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.
"1a, 25 (OH) 2D3" means la, 25-dihydroxyvitami-r~ D3.
"D3" means vitamin D3. The official IUPAC name for vitamin D3 is 9,10-secocholesta-5,7,10(19)-trien-3~i-ol.
"Transcaltachia" means the rapid hormonal stimulation of intestinal Ca2+ absorption.
"VDR" is a generic term that means 1a,25(OH)2Dj receptors that include VDRnu~ and VDRn,~".
"VDR""~" means nuclear receptor for la, 25 (OH) ZD3 interacting with 1a,25(OH)ZD3 or with the analogs of the invention.
"VDR~,~"," means membrane receptor for la, 25 (OH) 2D3 interacting with 1a,25(OH)2D3 or with the analogs of the invention.
"Ligand" means any small organic molecule that has a specific affinity for its cognate receptor. For example, .the ligand for the estrogen nuclear receptor is estradiol or its analogs. The ligand for the 1a,25(OH)ZD3 receptor, either VDRnu~ or VDR,~~" is la, 25 (OH) ZD3 or its analogs.

W0 99/1 6452 PC'TIUS98/1986z "PMSF" means phenylmethylsulfonyl fluoride.
"EGTA" means ethylene-bis(oxyethylenenitri~o)-tetraacetic acid.
"HEPES" means 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid.
"PKC" means protein kinase C.
"MAP-kinase" means mitogen activated protein kinase.
"Secosteroids" means compounds in which one of the cyclopentanoperhydrophenanthrene rings of the steroid ring 10 structure is broken. In the case of vitamin D3, 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 1a,25(OH)ZD3 or analog thereof 15 generated by interaction of 1a,25(OH)2D3 or analog thereof with the membrane receptor, that is observed within seconds to minutes following the exposure of cells to these compounds.
"Genomic response" or "slow genomic response" means a 20 biological response generated by interaction of "lcx,25(OH)2D3 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.
25 "DBP" means vitamin D binding protein.
"HRE" means hormone response element. Hormone response elements are composed of a specif is sequence of about 6-12 nucleotides in the promoter region of the specif is DNA constituting a gene which is regulated by 30 steroid hormone receptors, including the nuclear receptor for la, 25 (OH) ZD3.
"Target cell" means any cell in the body that possess either membrane receptors (VDR,~em) or nuclear receptors (VDRn"~) for loc, 25 (OH) ZD3.
3 5 BR ,FF _DF,~CRIPTION OF DRAjd, 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.
5 Figure 2 illustrates both the central role of receptors for 1a,25(OH)2D3 in mediating selective biological and the sites of action of both conformationally flexible and conformationally restricted analogs.
Figure 3 illustrates the conformational flexibility of 10 vitamin D molecules using 1a,25(OH)ZD3 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 1a,25(OH)2D3 or analogs to target cells.
Figure 5 represents a general model describing how 1a,25(OH)ZD3 and analogs of the invention, both conformationally flexible (Figure 5A and 5B) and conformationally restricted (Figure 5C), generate biological responses. ' Figure 6 illustrates mediation of the slow nuclear and rapid biological responses by 1a,25(OH)2D3 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 1a,25(OH)2D3 and selected analogs to the vitamin D-binding protein.
Figure 8 presents results of the binding of 1a,25(OH)2D3 and selected analogs to the nuclear receptor for la, 25 (OH) zD3 [VDR""~]
Figure 9 presents results of a classical in vivo biological assay in vitamin D-deficient chicks which quantitates the relative abilities of 1a,25(OH)2D3 and selected analogs to stimulate an intestinal Ca2+ absorption (ICA) and bone Caz+ mobilizing activity (BCM).
Figure 10 presents results from a cell culture assay which quantitates the relative abilities of 1a,25(OH)2D3 and the analog HS to stimulate cell differentiation.
Figure 11 presents results from a bioassay of transcaltachia, the rapid hormonal stimulation of 5 intestinal Ca2+ absorption, as stimulated by 1a,25(OH)ZD3 and selected analogs.
Figure 12 presents typical results from a cell culture assay which quantitates the relative abilities of 1a,25(OH)ZD3 and selected analogs to stimulate mitogen-activated protein kinases (MAP-kinase).
Figure 13 presents results from the assay of transcaltachia of the analog HL, namely 18,25(OH)ZD3, to inhibit the rapid response of stimulation of transcaltachia by la, 25 (OH) zD3.
15 Figure 14 illustrates the antagonist action of rapid responses elicited by treatment with 1a,25(OH)ZD3 and by analog HL.
Figure 15 illustrates the inhibition of activation of MAP-kinase medicated by 1a,25(OH)2D3, with analog HL present 20 at 10-9 molar concentration.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides analogs of 1a,25(OH)2D3 which are able to treat and ameliorate diseases and conditions connected with the vitamin D metabolism. These 25 analogs effectively control gene expression via slow genomic responses as well as rapid nongenomic cellular responses typically mediated by 1a,25dihydroxyvitamin D3 [1a,25(OH)ZD3]. The current invention, therefore, relates to novel biologically active analogs of 1a,25(OH)2D3. These 30 analogs are agonists of slow genomic responses or selective agonists or antagonists of rapid nongenomic cellular responses, depending on their chemical structures.
These analogs, their structures, their preparation and their chemical, physical and biological profiles are 35 described in the following Tables, Reaction Schemes and in Examples.

I . 1~,,, 25-Di~vdroxy vitamin D3 Analogs There are five groups of vitamin 1a,25(OH)ZD3 analogs which have the above described biological activity as agonists of the slow genomic responses or agonists or antagonists of the rapid nongenomic responses.
The group I is represented by compounds having a general formula I
R~
10 12 17___~..~

H

A

20 (I) wherein C1 and C3 are configurational isomers a and p which may be the same or different in a-a, ~i-~, a-(i or p-a configuration;
wherein C5-C6 double bond is cis or traps;
25 wherein C7-C8 double bond is cis or traps;
wherein C14 hydrogen is a or p;
wherein C16-C17 is a single or double bond;
wherein R1 is CH3 or CHZOH;
wherein R2 is a substituent selected from the group 30 consisting of substituents I-1 through I-10 H
.o '-'- ' ~_~ .5 _ - °H I ~ off 1 ~ ~ GH
lr S' H ~ H\ I.1 'OH
OH ' ._.;.t r' r' I_4 I_5 off ~~~ -H ~' ~~ H ~.~ .H OH

~..i°~~ ~° off OH
2 5 '~ ~'~~, J.r with the proviso that when R1 is CH3 and when C, and C3 are a-G, then RZ is not the substituent I-1, I-2, I-3, I-9 or I-10; or when C1 is in the a orientation and C3 is in the (3 orientation, C5-C6 double bond is cis or traps and C7-C8 double bond is traps, Rl is CH" C14 hydrogen is in the a orientation, C16-C17 is a single or double bond, then R2 is not the substituent I-1, I-2, I-3, I-4, I-5, I-9 or I-10;
SUBSTITUTE SHEET (RULE 28) or when C1 is in the ø orientation, C3 is in the ø
orientation, C5-C6 double bond is cis, C7-C8 double bond is traps, R1 is CH3, C14 hydrogen is in a orientation, C16-C17 5 is a single bond, then RZ is not the substituent I-1; or when C1 is in the a orientation, C3 is in the ø
orientation, C5-C6 double bond is cis, C7-C8 double bond is traps, R1 is CH20H, C14 hydrogen is in the a orientation, C16-C17 is a single bond, then RZ is not the substituent I-10 1; or ' when C3 is in the ø orientation, C1 is not hydroxyl, C5-C6 double bond is cis, C7-C8 double bond is traps, R1 is methyl, C14 hydrogen is in the a orientation, C16-C17 is a single bond, then Rz is a substituent I-7 or I-8; and 15 when C3 is in the ø orientation, C1 is in the a orientation, C5-C6 double bond is cis, C7-C8 double bond is traps, R1 is CH3, C14 hydrogen is in the a-orientation, C16 C17 is a single bond, then R2 is a modified version of side chain I-6 wherein the C22 methylene (CH2) is replaced by a 20 carbon-carbon triple bond.
The substituents I-1 - I-10 are the same as substituents II-1 - II-10, III-1 - III-10, IV-1 - IV-10 and V-1 - V-10. The designation I, II, III, IV and V show the group of the compounds having the general formula I, II, 25 III, IV or V to which the substituent selected form the substituents 1-10 is attached as R1, RZ or R,.

Compounds of the general formula I are prepared according to the Reaction Scheme 1.
"' ~2 R. R~, R, T ~ ~ 1 ' a) Smly THF.
0 7) 9uU.0'C PhCOCi PC(PPh~)a.
~.~~ iP:OH
t9 2) .i T HF. r, PaCCO H b) li .gin TSAF, T:~F
t ~
T?DV1S.'1'3 2 t v.TBCVIS 1 t C ~ D ' 10 3 ~ ~ / OH
t5 2 ~
au H ~i 4 O ett:er, 0-25 °C T90MS..'s~OT'eDMS OH
. o) hv.
450 watt a) Sp., R RZ ' Naphthalene- Hanovia lamp.
t Cr(CC)x, Guar, 5) hear. NaHCCy, acetone. 40 °C MeOH
BW anoi t If~ IH
tC) hv, 450waa t ~anovia iam~ I
9-aceytantnracene. 6 I
~ ~,,,~ pYrex.7~leOH ~ ~ H
HO'~~CH HO\~'CH
OOH
R, ~~ R RZ
OH
t ) SOZ ~
~ H t2) heat, NaHCOy ethanol ~ H
n 8 ~ 9 HO ~ H ~~
HO\~OH
___.___________ 8....__..__.'3j _.. R.;~'Z.____._.._.._. ___._.........

OTI H 11 a, X = TBDMS _W)_Ta:,F. Rt R2 T3DMSOr~ OTBDMS Pd(Poh~)2(OAc ~ 'f' 11 b, X = H ~ r OH
V ; "' Cul, EtZNH. DM~. rt \ ~ i51 He. Pd. Lindtar ~ qutnoune, nexanes I) H
XO~OX HO
_____._.__....._.__.__..__._.._..___.__.___._...._____...._.._______________.__ _... 12 T6) Iscoc;ane.
PhyP.O i rellux t7) n-Bull or PhLi. -73 °C. THF
13 ~ ta) R.~Rz I H

-aoMSO'~''o T aoMS .~ , C H 2 HOr~OH
?) T o,~F. T HF
SUBSTITUTE SHEET (RULE 26) R1 & R2 refer to the substituents of general formula (I) or their suitably protected forms (R'1 & R'2), usually as their silyl ethers; all structures may have single or double bonds across the C16-C17 positions.
5 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. The starting A-ring fragments 1 and 13, 20 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 10, wherein the substituents R'1 and R'2 are 15 the alcohol protected forms of R1 and R2 given in general formula I, are prepared as recently reviewed in Reviews, 95, 1877-1952, (1995). J. Org. Chem. 60, 6057-6061, (1995). J. Org. Chem., 58, 1895-1899,(1993). J. Orc~.
Chem., 54, 4072-4083, (1989).
2 0 In Scheme 1, general structures 6 , 7 , 8 , and 9 , with or without a double bond across C16-C17, are collectively represented by generic structure I.
In Scheme 1-A, the vitamin D A-ring fragment 1 is treated in step 1 with butyllithium and then the resulting 25 lithium salt is added to ketone 2 in step 2. The product from step 2 is reacted in step 3 with butyllithium and then benzoyl chloride to afford the propargyl benzoate 3.
Reduction of 3 with samarium iodide with appropriate additives as in step 4 followed by deprotection with 30 tetrabutylammonium fluoride (TBAF) in step 5 affords the 6~3-vinylallene analog 4. Photochemical irradiation as in step 6 affords the corresponding 6a-vinylallene analog 5.
Chromium(0) mediated isomerization of 4 in step 7 leads stereoselectively to the C5-C6 cis,C7-C8 cis analog 6, 35 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 il-12, respectively), to the corresponding C5-C6 traps compounds 7 and 9, respectively. Additional details for a specific case of the pathway of Scheme 1-A can be found in J. A_m__. Chem- sin 116, 6207-6216, (1994).
In Scheme 1-B, an alternative route to 8, and hence also 9 as in Scheme 1-A, starts with the palladium(0) mediated coupling of 1 with 10 in step 13 to afford lla.
Deprotection of the latter in step 14 to llb followed by Lindlar semi-hydrogenation (step 15) of the latter (llb) affords the previtamin type compound 12. Heating previtamin 12 at approximately 100°C (refluxing isooctane) in step 16 affords the desired 8.
In yet a third alternative Scheme I-C, the A-ring phosphine oxide 13, after deprotonation in step 17, is coupled with CD ring fragment 2 in step 18 (a so-called Horner-Wittig reaction). After deprotection in step 19, the resulting product is 8.
Table 1 lists subgroups of analogs falling within the scope of the Group I.
TABLE

= ~~s 1 Sc~. rs '.'~~-~ ~. ~~ 2 ~. g-?
a C 1 ~

$2:b5:~_a:~t5 I/1 a-a,- cis or cis aor single CH,or all with or a-,-a traps traps double CH,oH proviso I/2 -a cis traps a single CH, all I/3 -p cis traps a single CH, I-2,9,10 I/4 - cis traps a single Ca, analog H

I/5 a- cis traps a single CH,OH all I/o a-p cis traps a single CH:OH I-2,3,9,:0 T_/7 -~ cis traps a single CH_CH al'_ ,_'/8 - cis tra.~.sa single C ,OH T_-_2, 3, 9, 1:

!9 a-(i cis traas Q single Cri,orall 3 5 cH.oH

./10 a- c~.s traps single CH,or i-, 2, 3, 4, 9, 1C

C'.-:.OH

/11 -a cis traps p single C:i;oral.

suBSmv~ sH~r ~RU~ zs~

I/12 p-p cis traps p single CH,orI-,3,4,9,10 CH,OH

i/13 p-p cis traps a double CH, all 1/14 a-p cis traps a single CH;OHall I/15 p-p cis traps a single CH:OHall I/16 a-p cis traps p single CH,orall CHiOH

I/17 p-p cis traps p single CH~orall Z CH,OH
O

1/18 p-a cis traps a single CH, analogHH

I/19 a-p cis traps a single CHI analogHJ

I/20 a-p cis traps a single CH,OHanalogHS

I/21 a-p cis traps p single CHI analogGE

1/22 a-p cis traps a single CHI analogDE

I/23 a-p , cis traps a single CH, analogDF

I/24 a-p cis traps a single CH, analogHQ

I/25 a-p cis traps a single CH, analogHA

1/26 a-p cis traps a single CH, analogEv 2 1/27 a-p cis or cis a single CH, all traps or double I/28 a-p cis or cis a single CHI I-1 traps or double I/29 a-p traps cis a single CHI analogJS

I/30 a-p cis cis a single CH, analogJR

1/80 deoxy-p cis traps a single CH, analogJX

I/81 deoxy-p cis traps a single CHI analogJY

I/84 a-p cis traps a single CHI analogIB

30 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 35 8 (HL), Example 9 (HQ), Example 10 (HR), Example il (HS), Example 12 {IB), Example Z7 (JR), Example 18 (JS), Example 19 (JV), Example 20 {JW), Example 21 (JX), and Example 22 (JY).
SUBSTITUTE SHEET (RULE 26j 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 R1 is methyl, C1 hydroxyl is in configuration, C3 hydroxyl is in a configuration, C14 hydrogen is in a configuration and R2 is the substituent 2, 9, 10.
The representative analog is the analog HL.
The agonists of Group I are represented by a general 10 formula I wherein R1 is CH20H, C1 hydroxyl is in a configuration, C3 hydroxyl is in (3 configuration, C14 hydrogen is in a configuration and Rz are the substituents I-1 - I-10, preferably substituents I-2, I-3, I-4, I-9 and I-10.
In the same group, the antagonist are compounds wherein 15 R1 is CHzOH, C1 hydroxyl is in ~i configuration, C3 hydroxyl is in a configuration, C14 hydrogen is in a configuration and RZ
are the substituents I-1 - I-10, preferably substituents I-2, I-3, I-9 and I-10.
The group of agonists is represented by a general formula 20 I wherein R1 is CH3 or CHZOH, C1 hydroxyt- is in a configuration, C3 hydroxyl is in p configuration, C14 hydrogen is in p configuration and RZ are the substituents I-1 - I-10, preferably substituents I-1, I-2, I-3, I-4, I-9 and I-10.
The group of antagonists is represented by a general 25 formula I wherein R1 is CH3 or CH20H, C1 hydroxyl is in ~i configuration, C3 hydroxyl is in ~i configuration, C14 hydrogen is in p configuration and RZ are the substituents I-1 - I-10, preferably substituents I-1, I-2, I-3, I-4, I-9 and I-10.
The group II is represented by compounds having a general 30 formula II 1a R, --H
HO CH3 11 C 13 Ij 16 4 i5 3 4 5 ~ ~ (II) 35 HO B ~ H
s (II) wherein C1 and C3 are positional isomers a and ø which may be the same or different in a-a, ø-ø, a-ø or ø-a configuration;
wherein C9 hydrogen and CIO methyl are positional isomers 5 a and ø which may be the same or different in a-a, ø-ø, a-ø
or ø-a configuration;
wherein C16-C17 is a single or double bond;
wherein R1 is a substituent selected from the group consisting of substituents II-1 through II-10.
n " zf H
,o - ~s ,6 >>
OH '~OH
' ___.H ~, ' _.__H ' ....H
J,r f..t ~ ~. ~OH

H . H H .. \H
H
20 1 -.._H OH ._.H

II-a II-S
~ OH I ~ OH
..H __ _.~OH
~ i ~ T ~ _.
II-b \~OH O OH
3 0 '~ ~ ~ ~ . ..H

with the proviso that when C1 and C3 are a-ø , C9 and Clo are a-a, ~i-ø, a-ø and ø-a, and C16-C17 is a single bond, then R1 is SUBSTITUTE SHEET (RULE 26) not the substituent II-1.
Compounds of the general fonaula II are prepared according to the Reaction Scheme 2.
Scheme 2 A
) ~w I ~ h~ 2 ~ I
T3DMSO~OTBDhIS OTf ~'~ H 38, X - TBDMS
Pd(PPh~)zyOAe)3 . 3b, X = H CH
lQ ~ Cul, EtzNH. DMF, rt f~ ( 31 H,. PC. Lindlar ~mncune, nexanes XO OX HO

4) hv, 450 watt Hanavia lamp, Pyrex, MeOH 5) 150 °C, DMF, base, to hours Fiv R, R~ p, OH
O ( ~ ; OH ' OH
H I H_' + H H ' , ~ I
HO ~ HOr ~ I ~~H I H ~ I~H I H
h0 HO
5-9a,10p 5-9Q,10a 5_ga,t0a f 5-9p,10(i __.._____._..._.~..._..___._..__..___.____..._.._.________....R .._._..___.__-9) t~ °C DMF,__...._._._._..__..._..
base, 18 hcurs 2 0 PhzP=O
6) n-BuLi or PhLi. -78 °C, THF
6 ~ ~ n~ I H
~ ~ I 8 TBDMSO~OTBOMS
p H 7 HO OH
A) TBAF, THF
R1 refers to the substituents of generic formula (II) or the suitably protected forms (R'1),usually as the silyl ether;
all structures may have single or double bonds across the C16-17 positions.
30 Reaction Scheme 2 illustrates preparation of compounds of the Group II.
Compounds of the general formula II are prepared according to Scheme 2 using the two general approaches shown as Scheme 2-A and Scheme 2-B. The starting A-ring fragments 35 1 and 6, 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 SUBSTITUTE SHEET (RULE 26) Authors° of the Journal of Organic Chemistry), as well as the CD fragments 2 and 7, wherein the substituent R'1 is-the alcohol protected form of R1 given in general formula II, are easily prepared as recently described in Chemical Reviews.
95, 1877-1952, (1995). J. Orq. Chem., 60, 6057-6061, (1995).
J. Org~. Chem. , 58, 1895-1899, (1993) . ,J-Or~g. Chem. , 54, 4072-4083, (1989).
As indicated in Scheme 2, each compound may have a single or double bond across C16-C17. In addition, 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 palladium(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 as and ~3~3 isomers known as the pyrocalciferol and isopyrocalciferol types of vitamin D provitamins 5. By contrast, as shown in step 4, photochemical irradiation through pyrex us~.ng a medium pressure mercury lamp affords the 9a,10(3, and the 9p,10a provitamin D type isomers known as the dehydrocholesterol and the lumisterol analogs 5.
In a second alternative to Scheme 2-A, 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. After deprotection in step 8, the resulting product is 8 which, as described earlier, can be heated in step 9 at elevated temperatures to the same 9a,10a and 9p,10(3 provitamin D diastereomers 5, respectively.
Table 2 lists subgroups of analogs falling within the scope of the Group II.

Formula C1=C3 C9H-C10CH3 C16-C17 ~1 Substituents II/31 a-a,p-p aa,a[3, single all a-(i, p-a (3a, Qa double II/32 (i-p p-a single all 4 0 double II/33 p-p p-a single II-1,2,4,10 double II/39 p-p a-p single all double II/35 p-p a-p single II-1,2,9,10 double II/36 a-p a-a single all double II/37 a-p a-a single II-1,2,9,10 double II/38 a-p a-a single analog JO Check (II-I) II/39 a-p p-a single all 2 double II/40 a-p p-a single II-1,2,4,10 II/41 a-p p-a single analog JN (iI-1) II/42 a-p p-p single all double II/43 a-p p-p single II-1,2,4,10 3 double II/94 a-p p-p single analog JP (II-1) II/45 a-p p-a single all ' double II/46 a-p p-a single II-1,2,9,10 double 4 II/47 a-p p-a single analog JM (II-I) II/98 a-a p-a single II-1,2,9,10 double 4 II/49 a-a a-p single II-1,2,9,10 double II/50 p-a p-a single II-1,2,9,10 double The analogs listed in Group II are represented by the analogs identified as JM, JN, JO and JP.
These analogs, depending structure, have biological activity on their a as agonistsor antagonists genomicresponses or the rapid of slow 55 nongenomic s.
response In Group II, the antagonists are represented by the generic is in ~i configuration, formula II wherein C1 hydroxyl C3 hydroxyl configuration, is in C9 hydrogen ~3 is in (3 and C10 methyl the substituent II-1, is in a configuration and R1 is II-2, II-4 and II-10, preferably the substituents II-1, II-2, and II-10, or wherein C1 hydrogen is in (3 and C3 is-in ~i configuration, C9 hydrogen is in a and C10 methyl is in configuration and R1 is the substituent II-1, II-2, II-7, II-5 10, and is preferably the substituent II-1, IT-2 or II-10.
In Group II, the agonists are represented by the generic formula II wherein C1 hydroxyl is in a configuration, C3 hydroxyl is in p configuration, C9 hydrogen is in a and C10 methyl is in a configuration and R1 is the substituent II-1, 10 II-2, II-4, II-10, and preferably it is the substituent II-1, II-2, and II-13. The specific agonist of this group is the analog JO where R1 is the substituent II-1. Preparation of the analog JO is described in Example 6.
The other agonists of the Group II are represented by the 15 general formula II wherein C1 hydroxyl is in a and C3 hydroxyl is in (3 configuration, C9 hydrogen is in ~ and C10 methyl is in a configuration and R1 is the substituent II-1, II-2, II-4, II-10 and, preferably, it is the substituent II-I, II-2 and II-10. The specif is agonist of this group is the analog JN
20 where R1 is the substituent II-1. Preparation of- the analog JN is described in Example 5.
The other agonists of the Group II are represented by the general formula II wherein C1 hydroxyl is in a and C3 hydroxyl is in ~i configuration, C9 hydrogen is in ~ and C10 methyl is 25 in a configuration and R1 is the substituent IT-1 - II-10, preferably the substituent II-1, II-2, II-4 and II-10. The specific agonist of this group is the analog JM where R1 is the substituent II-1. Preparation of the analog JM is described in Example 5.
30 Still another agonists of the Group II are represented by the general formula II wherein C1 hydroxyl is in a and C3 hydroxyl is in p configuration, C9 hydrogen is in p and C10 methyl is in p configuration and R1 is the substituent II-1 -II-10, preferably II-1, II-2, II-4 and II-10. The specific 35 agonist of this group is the analog JP where R1 is the substituent II-1. Preparation of the analog JP is described in Example 6.

The group III is represented by compounds having a general formula III
78 Ri 2 A ~ 10 8 H
HO 3 a ~ 7 (III) wherein C1 and C3 are positional isomers a and ø which may be the same or different in a-a, ø-ø, a-ø or ø-a conf igurat ion;
wherein C14 hydrogen is a or ø;
wherein C16-C17 is a single or double bond;
wherein R1 is a substituent selected from the group consisting of substituents III-1 through III-10 -- I ON I OH
..4 =~ ..u ' _OH
ff ~ f.J' I
III-1 III-? III-3 ~, -.H H .H
'Z,~ '~ s ..rr 3 0 ~ o~
w .../~o a w a ~ ....
~., ...u ~~c ~! ...y ~ GH
~~_r .. . J" ..
III-6 III-i III-S
35 ,, o ~oh SUBSTITUTE SHEET (RULE 28) with the proviso that when C1 and C3 hydroxyls are in a-~i configuration, C14 hydrogen is a and C16-C17 is single bond, then R1 is not the substituent III-1, III-2, III-3, III-9, III-10; or when C1 and C3 hydroxyls are a-p and C14 hydrogen is a and C16-C17 is a single or double bond, then R~ is not the substituent III-4 and III-5.
Compounds of the general formula III are prepared according to the Reaction Scheme 3.
Scheme 3 A a.
~ 1 I , a..
I \ i ~ 1 H 3a. X = THD61S _2~ T3a~, T3DMS.~,~OT30MS ~~I 3b X o H
?C(P?h~~(CAc;,. . CH
Cul, E:=VH, DMF, rt I
I 31 H,. PC. lindlar Su,noana.naiaws I H
XO CX HO
._....._.._.__._B
......._.._....._......_____._...........__._.........._.._____............_...
..... 4....._._.....
R~
Ph=?.O p, ~1 n-a~u or ?hLi. -7>! °C. THF ~ ~ Dvss~Marsin Oxi'rstion ar 2 O MnO,-CH;C:, O
3 ~ 5) ~ , ( H
I 7 I ~ H
19DMS0~'OT20MS HO
I

6i CAF. i rig 9) NaBH(OAC)~, g) NaBHv.
M10H ~H
4 (C1.C3, 2 5 a-p or p-a) a (C1.C3, a-a or p-p) R1 refers to the substituents of generic formula (III) or 3o the suitably protected forms (R'11, 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.
35 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
SUBSTITUTE SHEET (RULE 26) 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'1 is the 5 alcohol protected form of R1 given in general formula II, are easily prepared according to references listed above.
As indicated above for Scheme 3, each compound may have a single or double bond across C16-C17. Thus, compound 4 is the same as the compound having general formula III.
10 Reactions illustrated in Scheme 3-A begins with the palladium(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.
15 In a second route, shown as scheme 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. The latter affords 7, which on selective allylic oxidation using either the Dess-Martin periodinane 20 oxidation method or the more classical manganese-dioxide in dichloromethane affords the previtamin ketone type 8 shown in Scheme 3B. On the one hand, normal sodium borohydride reduction in methanol affords the previtamin type alcohol 4 wherein the two A-ring hydroxyls at C1 and C3 are both cis to 25 each other, either a-a or ~i-p. In contrast, reduction of the same ketone 8 using sodium triacetoxyborohydride in methanol as shown in step 9, affords the alcohol 4 but stereoselectively in such a manner that the two hydroxyls at C1 and C3 are trans to one another, i.e. C1-C3 being a-p or 3o p-a.
Table 3 lists subgroups of analogs falling within the scope of the Group III.

Formula C1-C5C5 C~ C16C1? $~ S~,ibstituents III/51 aa, a(3 a or (i single all pa:aa double III/5.2 p-p a single all 4 0 double III/53 p-a a single III-1,2,4,7,9,1a double III/54 a-p a single all double III/55 a-p a single III-1,2,9,7,9,10 double IiI/56 p-p p single all double III/57 p-p p single III-1,2,9,7,9,10 double III/58 a-p p single all double III/59 a-p p single III-1,2,4,7,9,10 double III/60 a-p p single analog GF (III-1) 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.
In Group III, the agonists and antagonists are represented by the generic formula III wherein C1 hydroxyl is 30 in a or ~ configuration, C3 hydroxyl is in a configuration, C14 hydrogen is in a or ~ configuration, C16-C17 is a single or double bond and R1 is the substituent III-1 - III-10.
Preferred group of compounds of the Group III are compounds wherein C1 is in a configuration, C3 is in 35 configuration and the R1 substituent is selected from the group III-1 - III-10.
The specific agonist of this group is the analog GF where R1 is the substituent III-1. Preparation of the analog GF is described in Example 2.

The group IV is represented by compounds having a general formula IV
18 R~
---H
11 C i3 D17 _.
H

6 1~ OH

(IV) 15 wherein C1 and C3 hydroxyls are positional isomers a and p which may be the same or different in a-a, p-(3, a-~ or ~-a conf iguration;
wherein the C5-C6 is in a or (3 configuration;
wherein C14 hydrogen is a;
wherein C16-C17 is a single or double bond;--wherein R1 is a substituent selected from the group consisting of substituents IV-1 through IV-10 .0 '-= 2.t ~5 26 23 ~~OH ~ ~OH
.._.H 27 ~ ' I _...H ._.H ~
'OH

H .H H ..~H
H
.._H OH __H
15 ~' ~ .rr Iv'=~ Iv-s OH ~ OH
..H \ _.H _ ~ /
-~ ~OH
2 0 ~' ~ ~,,.r ~' ~ ~.r~' ~' .rte Iv-s Iv_~ Iv-s 0~~ H

OH
25 ~ ~ ~ ~ i . ._H

Compounds of the general formula IV are prepared a according to the Reaction Scheme 4.
SUBSTITUTE SHEET (RULE 26) 7 1 c) Smh. THF.
31 9uli: ~ Pd(PPh~i,. I
6 1) EuL. 0 °C PhCOCI ~ iPAH
c a "~~9 2., ~,~ THF, r, PhCGO N 5) T3aF, T ~F
~aoh,so=3~-oTaoMS ,~ 1~ 1 p~;a 3 ~~~ ~ °H
1 ~5 2 a O h e~'er, 0~25 °C T30MS0~'~v T 30h1S OH 4 (63) 6) hv, ~' ao watt Hanovia lamp, Guari., h'aOH
H
~ OH
'~ (fia, off R1 refers to the substituents of generic formula (IVj or the suitably protected forms (R'lj, usually as the silyl ether; all structures may have single or double bonds across the C16-C17 positions.
Rl refers to the substituents of generic formula (IVj or the suitably protected forms (R'lj, usually as the silyl ether; all structures may have single or double bonds across the C16-CI7 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 Schema 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 Chemistryj, as well as the CD fragment 2, wherein the substituent R'1 is the alcohol protected form of R1 given in general formula IV, are easily prepared as described in above cited references.
As indicated in Scheme 4, each compound may have a single or double band across C16-C17.
In Scheme 4, the vitamin D A-ring fragment 1 is treated in step 1 with butyllithium and then the resulting lithium sussnrurE sHE~r iRU~ 2s~

WO 99/16452 PCTIUS98/198b2 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. As similarly described in Scheme 1, reduction of 3 using samarium 5 iodide, catalytic palladium(0) 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 6a-vinylallene analog 4. Photochemical irradiation as in step 6 using a 450 watt medium pressure mercury lamp with methanol 10 as solvent affords the corresponding 6a-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.

C1-C3C3 CS-C6C6 C16-C17 $1 IV/61 aa-pp a or ~i single all a(3-pp double IV/62 a-a a single all double IV/63 a-a a single IV-1,2,4,7,9,10 2 5 double IV/69 ~p-p a single all double 3 0 IV/65 p-p a single IV-1,2,9,7,9,10 double IV/66 a-p a single all double IV/67 a-(i a single IV-1,2,4,7,9,10 double IV/68 a-~3 a single analog JW (IV-1) IV/69 (i-a a single all double IV/70 p-(i a single IV-1,2,9,7,9,10 4 5 double IV/71 a-a (3 single all double IV/72 a-a p single IV-1,2,4,7,9,10 5 0 double IV/?3 p-(3 (i single all double 5 5 IV/79 p-p p single IV-1,2,9,7,9,10 double IV/75 a-p p single all double IV/76 a-p p single IV-1,2,4,7,9,10 double IV/77 a-p p single analog JV (IV-1?
IV/78 p-a p single all double IV/79 p-a a single IV-1,2,4,7,9,10 double The analogs listed in Group IV are represented by the analogs identified as analogs JV and JW. 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.
In Group IV, the agonists and antagonists are represented by the generic formula IV wherein C1 hydrogen is in a or ~i configuration, and C3 is in a or ~i configuration, C5-C6 is in a or (3 configuration, C14 hydrogen is a, C16-C17 is a single or double bond and R1 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 C1 is in a configuration, ~3 is in p conf iguration and the R1 substituent is IV-1. The specif is agonists of this group are the analogs JV and JW.
The compounds of Group V are represented by a general formula V
---H
3 5 g ~ ~ (V) wherein C1 and C3 are positional isomers a and p which may be the same or different in aa, phi, a~i or pa configuration, wherein C5-C6 double band is cis and C7C8 double band is 4 5 trans ;

WO 99/16452 PGTlUS98/19862 wherein C16-C17 is a single or double bond; and wherein R1 is a substituent is selected from the group consisting of substituents V-1 through V-10 H
' =0 ° .3 =1 =5 ~H O ~ ?:
__ __H --H
1r~ ~ it ~ pH
10 ~~f''~ .rr' 'r'' V.~ V-3 H . H H .. \H
H
--H ~H ' ---H
15 '~ t r .rr ~' ~ .rr' V
OH OH
..H I .._H~OH
~,~r~. 'Z, ' ..rr - '2' ~ ~rr 2 0 V.6 'r.~ V.8 O H OH
~OH O
~ ~ t .._.H
25 v.9 v-io Compounds of the general formula V are prepared according 30 to Reaction Scheme 5 using the two general approaches shown in Scheme 5-A and Scheme 5-B.
SUBSTITUTE SHEET (RULE 26) A
5 ~~ ,) ~ I "' I z ~ x O T t I I '~ Sa~ X c TBOhI~ 21 T2AF. R.
T 3ChlSv' v _O ~ oCVIS ~J T eiF
vaiaQh~~roac),. 3b, X ~ Ii off c~l. ~cwH, ow, r; ~ I a, H,. va. I~a~r ~u,ncuna.ntaants I
XO'~ OX YOr ...............8..............................................F
......................
a) Isooc;are.
?h P~O
10 ~ 3) n-duLi or Phli. ~i3 °C. THF nAux 6 I 6) n , TaoMSO orao~,s O NO OH
~ TdAF. THF
R1 refers to the substituents of generic formula (V) or the suitably protected forms (R'1), usually as the silyl ether. The starting A-ring fragments 1 and 6, wherein the C1-C3 alcohols are masked as their TBDMS ether protecting groups 20 as well as the CD fragments 2 and 7, wherein the substituent R'1 is the alcohol protected form of Rl given in general formula V, are prepared according to Chemical Reviews. 95:
1877-1952 (1995). ~-,Org. C] em., 58: 1895-1899 (1993);
Org. Chem., 54: 4072-4083 (1989) as cited above. Each 25 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 30 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. In an alternative scheme, namely Scheme 5-B, the A-ring phosphine oxide 6 is directly treated with strong base as shown in step 35 5 whereupon Horner-Wittig reaction with ketone 7 produces a protected triene as given in step 6. Deprotectian of the resulting product with TBAF and tetrahydrofuran in step 7 of Scheme 5 also affords the same analog 5.
SUBSTITUTE SHEET (RULE 28) Formula C1-C3 Hi V/82 aa-pp all ap-pa V/ a-a all V/ a-p all 1 0 V/83 a-p analog LO (V-1) V ~-a all V a-Q all A representative analog of this group is analog LO which is an agonist of slow genomic and rapid nongenomic responses.
II . Bio1_ocr; c-a1 Ac ~; i y of i ry~~~OH) ~R3 An310as I . MOde of Acti nn of ~i tami n 1'7 A. Vitamin D
Vitamin D is essential for maintenance of calcium/mineral homeostasis. One of the _ vitamin D
metabolites, namely 1a,25(OH)2-vitamin D3 [1a,25(OH)2D3] 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 la, 25 (OH) 2D3.
1a,25(OH)2D3 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.
B. yitamin D Endocr;nP ~yato", The scope of the biological responses related to vitamin D is best~understood through the concept of the vitamin D
endocrine system model as seen in Figure 1.

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 target organs. As seen in Figure 1, photoconversion of vitamin D (7-dehydrocholesterol) to vitamin D3 (activated 7-dehydrocholesterol) occurs in the skin. Vitamin D3 is then metabolized by the liver to 25(OH)D3. The kidney, functioning as an endocrine gland, converts 25 (OH) D3 to la, 25 (OH) 2D3 and 24R,25(OH)ZD3. The hydrophobic vitamin D and its metabolites, particularly 1a,25(OH)2D,, are bound to the vitamin D binding protein (DBP) present in the plasma and systemically transported to distal target organs, as seen in Figure 4.
la, 25 (OH) 2D3 binding to the target organs cell receptors is followed by the generation of appropriate biological responses through a variety of signal transduction pathways.
Figure 2 presents a more comprehensive version of the vitamin D endocrine system specifically indicating selective generation of biological responses by the analogs of la, 25 (OH) 2D3 resulting in the treatment of specif-ied disease states. A detailed tabulation of the cells containing the nuclear receptor [VDR""~] for la, 25 (OH) 2D3 as well as an enumeration of the tissue location of the membrane receptor [ VDR,~e",] where rapid response is initiated are seen in the lower part of the Figure 2.
Figure 2 additionally shows the target sites for application of 1a,25(OH)2D3analogs functioning as agonist and antagonist.
C. Co_n_fo_rmat,'_onal F1 exi hi 1 i~y of Vitami r, D Seco Steroids 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 flexibility is seen in Figure 3.
In biological systems, there are a multitude of shapes of -1a,25(OH)ZD3 available to interact with receptors to generate biological responses. Different shapes of 1a,25(OH)2D3 are recognized via different ligand binding domains present on the VDR"u~. VDRme"" and DBP. A variety of analogs- of la, 25 (OH)ZD3, some of which are as conformationally flexible as 1a,25(OH)2D3 and some of which are conformationally restricted, such as, for example, the family of 6-s-cis locked analogs, were synthesized and tested.
Figure 3 illustrates the conformational flexibility of vitamin D molecules using 1a,25(OH)ZD3 as an example. Figure 3A shows the dynamic single bond rotation of the cholesterol-like side chain of 1a,25(OH)ZD~, 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.
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 la-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-traps) conformation of 1a,25(OH)2D3.
Conformationally flexible analogs of 1a,25(OH)ZD3 as seen in Figure 3 , can interact with both the VDR"uc and the VDRn,~"
while 6-s-cis locked conformationally restricted analogs interact only with the VDR~", A tabulation of the analogs of the invention, their conformational flexibility and general biological properties are presented in Table 6.

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SUBSTITUTE SHEET (RULE 26) O. V;tami_n D-bind ny Pry Vitamin D binding protein (DBP) 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 5 played by its metabolites the DBP in transporting both 1a,25(OH)2D3 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 10 D-binding protein (DBP) in transporting 1a,25(OH)2D3 or its analogs throughout the circulatory system.
As seen in Figure 4, DBP either binds 1a,25(OH)ZD3 as it is secreted by the kidney or binds analogs at their site of the encounter following the analog administration. For example, 15 when the analog is administered orally, the DBP binds it after its intestinal. absorption. After intravenous administration, DBP binds to the venously administered and available analog in the circulating blood. Without the intervention and transport by DBP, the relatively water insoluble analogs would not find 20 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 (VDR~",) or nuclear receptors (VDR""~) for 1a,25(OH)ZD~. The DBP bound to the analog moves through the circulatory system and makes the bound analog universally 25 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""~ and VDR"" receptor 30 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 la, 25 (OH) ZD, or analogs governed by the equilibrium constant or affinity for ligand binding by DBP. The important consequence is that there are low 35 concentrations of free analogs distributed, throughout the circulatory system which are available for uptake by target cells and interaction with the VDR~"~ and/or VDR~.
As shown in Figure 4, the DBP has the capability to transport the conformationally flexible 1a,25(OH)2D~, suesnTUrE sH~r (RmE zs~

conformationally flexible analogs and 6-s-cis conformationally restricted analogs.
E. Mode-of-Act i nr, o a 5 (,CDs and Its Any 1 n~
The spectrum of biological responses mediated by the hormone 1a,25(OH)ZD3 occurs as a consequence of the interaction of 1a,25(OH)2D3 with two classes of specific receptors. These receptors are identified as the nuclear receptor, VDR""~ and the cellular membrane receptor, VDR"",. The VDR""~ protein contains a ligand binding domain able to bind with high affinity and with great specificity ia,25(OH)2D3 and closely related analogs.
1a,25(OH)2D3 has been found to generate biological responses via interaction with a putative membrane receptor [VDR"~"] which is coupled to cellular signal transduction pathways. This interaction generates rapid response via opening voltage gated 15Ca2~ channels and C1- channels as well as activating MAP-kinases. Different shapes of the conformationally flexible 1a, 25 (OH) 2D3 or its analogs bind to the VDR""~ and VDR,~" and initiate biological responses via activation of signal transduction mechanisms which are coupled to either the VDR""~ or the VDR""",. Thus the totality of biological responses mediated by 1a,25(OH)2D3 or its analogs represents an integration of both nuclear receptor and membrane receptor initiated events.
In terms of analogs of 1a,25(OH)2D~, there are two general classes of such analogs. There are agonists that generate responses similar to 1a,25(OH)2Dj and there are antagonists that block or minimize the responses initiated by 1a,25(OH)2D3 or agonist analogs. Further, agonist or antagonist molecules can either be fully conformationally flexible, like the natural hormone 1a,25(OH)ZD3 as seen in Figure 3, or be conformationally 30restricted. One example of a conformationally restricted agonist molecule is is,25(OH)2-7-dehydrocholesterol, analog JM, that is permanently locked in the 6-s-cis shape.
A detailed list of the conformationally flexible and restricted agonist and antagonist analogs is presented in Tables 6-8. Conformationally flexible analogs can interact with both VDR""~ and VDR",~. In contrast, 6-s-cps conformationally locked analogs can only interact with VDR,~",. The general mode of action by which 1a,25(OH)2D3 generates biological responses in SUBSTITUTE SHEET (RULE 28) target cells is shown in the three panels of Figure 5. The model seen in Figure 5 invokes ligand domains for receptors (the VDR""~ and WRY") with different specificities for different shapes or conformers of la, 25 (OH) ZD3. From the point of conformational flexibility, there exists two general classes of analogs. One class are those analogs that have complete flexibility around the 6,7 carbon-carbon bond, as does 1a,25(OH)2D3. The second class are those analogs which are conformationally restricted, such as 6,7-locked analogs. An example of such analogs are 1a,25(OH)2-7-dehydrocholesterol (JM) or 1a,25(OH)Z-lumisterol (JN).
Figure 5 compares the mode of actions of these two types of analogs, namely conformationally flexible analogs and conformationally restricted 6-s-cfs analogs. As seen in Figure 5A, 1a,25(OH)2D3 which is conformationally flexible interacts with both the membrane receptor depicted as VDR,o,e, located in the cell membrane, and with the cell nuclear receptor depicted as VDR""~ located in the cell nucleus of the target cell. The slow genomic responses appear after 1a,25(OH)2D3 or its analogs interaction with VDR""~. Rapid responses are generated upon interaction of la, 25 (OH) ZD, or its analog with VDRm~".
Conformationally flexible analogs of the invention, illustrated in Figure 5B, act similarly to 1a,25(OH)ZD3 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""~ and VDR",~.
In Figure 5C, where the action of conformationally restricted 6-s-cis analogs is illustrated, the only interaction which is observed is between the analog and VDR",~, receptor thereby resulting solely in selected rapid nongenomic biological responses.
Figure 6 represents a model and a description of the mechanisms of action by which 1a,25(OH)2D3 generates biological responses in target cells. As indicated at the top of Figure 6, the conformationally flexible natural hormone, 1a,25(OH)2D,, and conformationally flexible analogs interact with both the ~Rnuc and VDR,~. However, 6-s-cis locked analogs can interact only with the VDR"~",. After occupancy of the receptors by their SUBSTITUTE SHEET (RULE 26) ligand, appropriate signal transduction systems are initiated which ultimately lead to the generation of biological responses.
The bottom panel of the Figure 6 lists certain target cells for 1a,25(OH)2D3 and identifies typical responses of these cells to administration of 1a,25(OH)ZD3 or the analog which occur there.
Disease states for treatment with analogs of 1a,25(OH)2D3 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-cis locked analogs, that bind to the VDR~,m to initiate the generation of rapid biological responses. Occupancy of the VDR~~~ can lead to activation of a variety of intracellular messengers, such as cyclic AMP, protein kinase C, or increases in intracellular Ca2' concentration, which, depending upon the cell type, can cause the opening of calcium channels, chloride channels, or activation of mitogen-activated protein kinase.
In cells that have a VDRA"" linked to a calcium channel, there is an increase in Ca2+ ions moving into the cells that results in an increase in intracellular Ca2' concentrations. In intestinal cells, this will activate the rapid response of transcaltachia and increase the absorption of dietary Ca2+ into the body. In bone-forming cells (osteoblasts), opening of the calcium channel followed by the intracellular calcium increase results in increased activities of the osteoblasts on bone formation. Similarly, in pancreatic 8 cells, opening of calcium channels participates favorably in the processes governing the secretion of insulin.
In cells that have a chloride channel linked to a vDR~"
there is an increase in chloride ions which is known to be linked to water uptake by the cell leading to a condition of volume expansion. This chloride channel activation in osteoblast cells leads to increased activities in the osteoblast in bone formation. Dysfunction of chloride channel opening in kidney cells has been linked to x-linked hypercalciuric nephrolithiasis.
In cells that have the vDR",~ linked to activation of MAP-kinase, so called "message cross-talk" between the rapid response pathway and the nucleus results upon activation of MAP-SUBSTITUTE SHEET (RULE 28) kinase with analogs of the invention. The cell where VDRm,~ is activated resulting in rapid responses utilizes cross-talk between the membrane and the VDR"u~ receptor leading to modulation of gene transcription, seen in the center of Figure 5. The MAP-kinase activation leads to changes in the phosphorylation state of the proteins participating in the transcription complex, including the VDR""~. Then, depending upon whether the gene subject to regulation by the VDR~",~ is subject to up-regulation or down-regulation, there can be 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 processes to the overall outcome biological response for a variety of target cells. In turn, 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.
2 0 I I I . TheraneLt-. i_ca 1_ 1 y Act i vA Ana ~ ngs of la ,~( OH 1 ~~3 A. Classes of Analogs 1. 8~,g~
(a) ~onformat~ onal 1 y Flex; h1 A Genomi r ,a~~n~~_~t Anal nr~~
Conformationally flexible genomic agonist analogs are the analogs which interact with the nuclear receptor for 1a,25(OH)2Dj VDR""~ and are, therefore, involved in the slow genomic responses. Exemplary analogs in this group are analogs listed in Table 7.
In all categories, a two-letter code name for analog chemical identification is designated followed by the chemical name.
--DE 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor-la(OH)D3 DF 22-(p-hydroxyphenyl)- 23,24,25,26,27-psntanor -1a(O8)D3 SUBSTITUTE SHEET (RULE 26) WO 99116452 PCfIUS98/t9862 EV 22-(m-(dimethylhydroxymethyl)phenyl)-23,24,25,26,27-pentan or -la(OH)D3 GE 14-epi-la, 2 5 ( OH ) ZD3 5 Gf 14-epi-1a,25(OH)2-prs-D3 HJ 1a,25(OH)Z-3-epi-D3 HQ (22S)-1a,25(OH)2-22,23-diems-D~

HR (22R)-1a,25(OH)Z-22,23-diems-Dj HS 1a,18,25(OH)~D3 15 IB 23-(m-(Dimethylhydroxymethyl)phenyl)-22-yns-24,25,26,27-tetranor-la(OH)D~

JR la, 2 5 ( OH ) Z-7 , 8-c~.i-D3 JS la, 25 (OH)Z-5, 6-trams-7, 8-c~ta-D~

JV (1S,3R,6S)-7,19-retro-1a,25(OH)ZD;

(1S,3R,6R)-7,19-retro-1a,25(OH)ZD3 2 JX 22-(p-hydroxyphenyl)-22,23,24,25,26,27-pentanor-Dj JY 22-(m-hydroxyphsnyl)-23,24,25,26,27-pentanor-D3 LO 14a,15a-methano-1a,25(OH)ZD~

(bj Cenformatfonal 1 y Res r; c-te.a rpn~,mic A~oni at nab ~arss Conformationally restricted genomic agonist analogs are the analogs which bind with a specificity to the vitamin D nuclear 35 receptor vDR""~ and are therefore also involved in genomic SUBSTITUTE SHEET (RULE 28) responses.
(c) ~nformational~y Fiexibl,~ Nonq~enom~AQonWt a."i..,.a ~enerating~D;d Resbonse Conformationally flexible agonist analogs of la, 25 (OH) 2D3 which stimulate rapid nongenomic responses via interaction with the vitamin D membrane receptor VDR~~, are listed in Table 8.
Table 8 22-(m-hydroxyphenyl)- 23,29,25,26,27-pentanor-la(OH)D, DE

DF 22-(p-hydroxyphenyl)- 23,29,25,26,27-pentanor -la(oH)D, EV 22-(m-(dimethylhydroxymethyl)phenyl)-23,24,25,26,27-pentanor-la(off)D, GE 19-epi-la, 25 ( OH ) ZD, GF 14-epi-1a,25(OH),-pre-D, HJ la, 25 (OH) ~-3-epi-D, HQ (22S)-1a,25(OH)Z-22,23-diene-D, HR (22R)-1a,25(OH)z-22,23-diene-D, HS la,18, 25 (OH),D, IB 23-(m-(dimethylhydroxymethyi)phenyl)-22-yne-24;25,26,27-tetra nor-la(OH)D, JR la, 25 (OH),-7, 8-cis-D, JS la, 25 (OH) z-5, 6-traps-7, 8-cis-D, JV (1S,3R,6S)-7,19-retro-1a,25(OH)sD, SUBSTITUTE SHEET (RULE 26) ,'lye(1S,3R,6R)-7,19-retro-1a,25(OH)zD~

22-(p-hydroxyphenyl)-22,23,24,25,26,27-pentanor-D, Jy 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor-D~

I,p14a,15a-methano-la, 25 (OH) zD, (d) rnnf~rmat~nr~ Restricted Nonr~enomic Aqonist Conformationally restricted agonist analogs which generate nongenomic rapid responses via interaction with the membrane receptor for 1a,25(OH)2D, are listed in Table 9.
Table 9 JM 1a,25(OH)2-7-dehydrocholesterol JN 1a,25(OH)Z-lumisterol3 JO la, 25 (OH) 2-pyrocalciferol3 JP 1a,25(OH)2-isopyrocalciferol3 2.
(a) ~~nformationally Flexible Antagonists of Ranid Conformationally flexible antagonist of genomic responses function as antagonists of the vitamin D nuclear receptor.
(b) Conformationallv Restricted Antagonists of Ranid Conformationally restricted analogs which function as antagonists of nongenomic rapid responses via interaction with the membrane receptor for 1a,25(OH)2D3 are listed in Table 10.
SUBSTITUTE SHEET (RULE 26j l~. 25 (OH) 2-3-epi-D3 1D,25(OHy2D~

(c) ~onfo ate ona» v Restr~ cted Antag~on~'_sts of Rat~id Rp~monses Conformationally restricted antagonists of rapid responses function as antagonists of the VDR"a".
IV, B; o~ oQ~ cad Prod ~ p of 1a. 25 lOHl3~" AnaloQS_ A, Ana~oa Binding to the Vitamin D-Binding Protein 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.
It is therefore, important to determine the degree of binding of each analog to the DBP.
Analog binding to the DBP is illustrated in Figure 4 which su~rizes the key role played by the vitamin D binding protein in the transport of 1a,25(OH)2D3 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 (DBP) 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 1a,25(OH)2D3.
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 of f inity of binding of the analog to the DBP binding site is measured and expressed as Relative Competitive Index.
The more available a ligand is for uptake by a target cell, the more likely it is to interact with either the VDRt,u~ or the VDR"~, so as to generate biological responses.
The Relative Competitive Index (RCI) of several analogs of the invention is seen in Figure 7.
Figure 7 shows results of the determination of the RCI for SUBSTITUTE SHEET (RULE 26~

WO 99/16452 PCTlUS98/19862 representative analogs for the vitamin D binding protein (DBP) compared to 1a,25(OH)2D" identified as compound C. The compared analogs are 14a,15a-methano-1a,25(OH)2D3 (LO), 22(m(dimethylhydroxymethyl)phenyl)-23,24,25,26,27-pentanor-la-OH-D3 (EV) and (22R)-1,25(OH)2-22,23,diene-D3 (HR), all conformationally flexible genomic agonists. The RCI values expressed as (% maximum bound)-1 x 100 of the analog in competition with 1,25(OH)ZD3 are indicated in the Figure 7. By def inition the RCI for la, 25 (OH) 2D3 is set to 100% . The data seen in Figure 7 represent the mean of three determinations.
The results seen in Figure 7 indicate that compared to 100%
binding of 1a,25(OH)2D3 (C) to the DBP, analog LO binds to DBP
60% as tightly while analogs EV and HR bind only 25% and 48% as tightly to DBP. From the perspective of DBP functioning i.n vivo or in being present in the culture media used to nourish cells grown in tissue culture, analogs which have an RCI lower than 1a,25(OH)ZD, have a higher free concentration in solution and are more available for uptake into target cells. Conversely, analogs with an RCI for DBP greater than 100% ( la, 25 (OH) 2D;) , have a lower free concentration and are less available for uptake into potential target cells.
In terms of analogs relevant to this patent application as listed in Table 11, below, analog JX has the highest RCI for DBP, a value of 211,000 or 2110 times greater than the reference 1a,25(OH)2D3. This analog, therefore, binds very tightly to DBP
and has a much lower free concentration and lower availability for uptake by target cells. Conversely analog HL has an RCI of only O.I, which is 1000 times lower than that of the reference 1a,25(OH)ZD3. Thus, 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.
B. Biola$ical Evaluation of 3. 25 IOHI ~j~
Table 11 summarizes the biological evaluation of all the analogs of 1a,25(OH)2D3 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 SUBSTITUTE SHEET (RULE 25) as RCI), binding to the nuclear 1a,25(OH)ZDs 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 Ca2~ absorption 5 (ICA) and bone Ca2' mobilizing activity {BCM) determined in v3vo 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.
As seen in Table 11, twenty three analogs and 1a,25(OH)2D3 10(designated by analog code as C) were submitted to testing as outlined in Table 6. Of these analogs 22 are agonists, that is compounds which possess affinity for the receptor and are capable of combining with 1a,25{OH)2D3 receptor. one of the analogs is an antagonist (HL), that is a compound which does not 15 bind to the recegtor and in fact it blocks or inhibits the action of agonist for rapid responses.
Nineteen of the analogs are able to elicit both the genomic and rapid responses.
Four of the analogs (JM, JN, JO and JP) are able to elicit 20 solely rapid responses, that is to bind only to the membrane vDR~", receptors. The three of four analogs identified as eliciting the rapid responses show transcaltachia activity corresponding to about 50 to 60% of the 1a,25(OH)xD3 transcaltachia activity. Analog JN shows i0b% of binding to 25 vDR,~", receptor, that is, it has binding affinity higher than la, 25 {OH) 2D3.
Thirteen analogs (EV, GE, GF, HQ, HR, JM, JN, JO, JP, JR, JS, Jv and ?~0) have DBP binding activity lower than 1a,25(OH)2D3.
Consequently, these analogs are more available in their free 30 form in the circulating blood and are therefore more available for uptake by the target cell and more active in treatment of vitamin D diseases than 1a,25(OH)2D3.
Regarding binding to the nuclear receptor to elicit genomic responses, all tested analogs have lower binding affinity for 35 1a, 25-D receptor than la, 25 {OH) ZD3. Only the analog LO shows similar binding activity (98%) to that of 1a,25(OH)ZD3, followed by the analogs EV (62%), HR (52%), DE (29%), HS {25%), HJ (24%) and GE (15%). These analogs are therefore suitable for SUBSTITUTE SHEET (RULE 26) treatment of diseases where the slower genomic responses via gene expression are involved. For elicitation of classic vitamin D responses ICA and BCM, the best analog identified by its comparative activity with 1a,25(OH)2D3 is the analog LO, showing 30% of ICA and 50% of BCM, compared to 1a,25(OH)2D3.
All analogs disclosed herein having either genomic or rapid response or both are useful and suitable for treatment of diseases treatable with 1a,25(OH)2D3.
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~ r-1 N N f'~f M d' SUBSTITUTE SHEET (RULE 26) V. Genomic Res,,g A. Interaction of Ana'1 ~~_ ~/ ~f1 ~10f!pTl A mode-of-action and interaction of 1a,25(OH)ZD~ and the analogs of the invention with the VDRn"~ and VDR,~ to generate various biological responses is outlined in Figures 5 and 6.
After transport and delivery of 1a,25(OH)2D3, or the analog of the invention by DBP through the circulatory system, the 1a,25(OH)2D3, or the analog is disassociated from the DBP. The la, 25 (OH) 2D" 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""~ and VDR"",. As shown in Figure 5, panel A, the conformationally flexible 1a,25(OH)2D3, or the analog, then interacts either directly with the VDRm", 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""o .
Because of the high affinity of the VDR""~ for conformationally flexible analogs of 1a,25(OH)ZD,, 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. There is the general relationship between one gene, one mRNA molecule, and one specific protein. The specific protein then is involved in a critical way in elicitation of the biological responses which are governed by SUBSTITUTE SHEET (RULE 28) WO 99/16452 PG"f/US98I19862 the initiator of its biosynthesis, in this example, the VDR""
forming a complex with its hormone or analog ligand.
Thus, the occupied VDRnu~ 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). When a Vt?Rr",~ finds a specif is gene with a VDRE, then there ensues the formation of an active transcription complex.
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.
There are two general categories of WRE. 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. Thus, the specific presence of a conformationally flexible ia,25(OH)ZD3 (Figure 5A) or analog (drug) (Figure 58) in the target cell where there is a VDR"uc results in a change, either an increase or a decrease, in the production of specific messenger RNA molecules linked ultimately to the production of a specific biological response, as illustrated in Figure 6, left side.
The critical contribution of the conformationally flexible 1a,25(OH)2D3 or analog. (drug), is to regulate the gene transcription process. The resulting pool of messenger RNA
molecules is then translated resulting in either increased or decreased amounts of specific new proteins. These new proteins then engage in their regular function that varies depending upon the nature of the specific gene from which it was transcribed.
Genes that are turned-on by VDR""~/analog complex result in generation of specific proteins depending on the target tissue.
B. yp$"".. Rel_ati_vp Comyetitivp TTIf~PY As Assav The ability of analogs to mediate genomic responses are directly determined by the ability of the analog in question to bind to the nuclear receptor for la, 25 (OH) 2D3 [VDR""~1 ~ This SUBSnTUTE SHEET (RULE 26) ability is detected by the assay measuring Relative Competitive Index (RCI). Exemplary illustration of the RCI assay and results of RCI is seen in Figure 8.
Figure 8 shows Relative Competitive Index (RCI) 5 determination for representative analogs that bind to the nuclear receptor for la, 25 (OH) 2D3 [VDR""~] . The assay is based upon the principles of a steroid competition assay. A fixed amount of [3H] la, 25 (OH) 2D3 is mixed with increasing amounts of competitive analogs or the natural hormone, 1a,25(OH)2D,, and 10 incubated with a VDR""~ receptor preparation from chick intestine mucosa. The results are presented for 1a,25(OH)ZD3, analog LO
[14a,15a-methano-1a,25(OH)2D3] (1), analog HS [1a,18,25(OH)3D3) (x), and analog DF [22-(p-hydroxyphenyl)-23,24,25,26,27-pentanor-la-(OH)D3](D).
15 The results of Figure 8 indicate that analogs LO, HS and DF
bind 98%, 25%, and 5%, respectively, to the VDR""~ present in chick mucosa, compared to 100% binding of 1a,25(OH)2D3. These results indicate the relative ability of these particular analogs to regulate gene transcription through their binding to 20 the VDR""~. From these results, it is clear that analog LO is as active in generating nuclear responses as is the 1a,25(OH)2D3.
RCI of other analogs is shown in Table 6.
C. rn ; na1 ca1_c? mm Abso ntson and Bone Calcite Mobsli2ation Assavs 25 A primary fundamental physiological property of vitamin D
and particularly 1a, 25 (OH) ZD3 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) 30 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 Ca2+ absorption.
A second important physiological action of 1a,25(OH)2D3 is its effects on bone cells. Under circumstances of a dietary 35 shortage of calcium, the blood concentration of Ca2+ falls and the individual becomes hypocalcemic. In order to prevent an SUBSTITUTE SHEET (RULE 26) extreme reduction in the blood concentration of Ca2+, the organism utilizes 1a,25(OH)ZD3 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 tasted analog to mobilize bone calcium.
The natural hormone 1a,25(OH)2D, is very potent in the BCM assay.
For example, when 1a,25(OH)ZD3 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 Ca2+ mobilization.
Results of the testing of the analogs of the invention 3n vivo by the ICA and IBM assays are shown in Figure 9 which illustrates the capability of analogs LO, EV and HR to stimulate intestinal Ca2+ absorption (ICA) and bone Ca2+ mobilizing activity (BCM). In this study, the analogs of 1a,25(OH) were given i.m. to vitamin D-deficient chicks 12 hours before the assay began. The activity produced by 100 pmol of 1a,25(OH)ZDj was set to be 100% for both ICA and BCM. The dose of the analogs required to achieve a biological response for either ICA
or BCM equivalent to the 100 pmol dose of 1a,25(OH)2D3 was calculated and converted to a percentage. Results are expressed as mean t 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 D3 (+D3) was present in 3.25 nmol. The difference between the -D and +D3 groups was significant at P<0.01. ia,25(OH)2D3 and analogs LO [14a,15a methano-1a,25(OH)2D3], Ev [22-(m(dimethylhydroxymethyl)phenyl) 23,24,25,26,27-pentanor-la-OH-D,] and HR [(22R)-1,25(OH)2-22,23 diene-D3] were administered in 0.0065, 0.065, 0.65 and 6.5 nmol as shown.
As illustrated in Figure 9 and summarized in Table 6, the most potent stimulator of ICA and BCM was the reference compound SUBSTITUTE SHEET (RULE 28) 1a,25(OH)2D3. The comparative activity values expressed as percent of 1a,25(OH)ZD3 for both ICA and 8CM assays, as seen in Figure 9A(ICA) and Figure 9B(BCM), respectively, for each analog was as follows: analog LO (30%/80%), analog EV (30%/8%), and analog HR (12%/0.6%).
Table 6 shows ICA and BCM data for the analogs seen in Figure 9 as well as other analogs of the invention. For example, analog LO which has the highest ICA (30%) and HCM (80%) relative to the ICA and BCM values for 1a,25(OH)2D3 would be a highly effective stimulator of bone Ca2~ mobilizing activity (BCM) and reasonable stimulator of intestinal Caz' absorption (ICA) and is therefore useful for treatment of hypocalcemia and rickets. Additionally, analogs DE and EV show stimulating activity in both ICA and BCM assays.
C. Dell D?fferent,'_atson Aseav One of the recently discovered properties of the natural hormone 1a,25(OH)2D3, 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 1a,25(OH)2D, are dependent upon the widespread tissue distribution of receptors, both the VDR""~ and VDR~"" as described in Figure 2.
1a,25(OH)ZD, 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 1a,25(OH)2D3 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 1a,25(OH)2Dj is determined to be 1. If the analog has ED-50 of SUBSTITUTE SHEET (RULE 2B) 0.1, it achieves 50% of its maximal cell differentiation effect at a concentration of about one tenth that of la, 25 (OH) 2D3 and is, therefore, ten times more effective.
Figure 10 is dose-response of analog HS or loc, 25 (OH) ZD3 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 vitro blue tetrazolium (NBT). Each point represents the mean of two experiments with triplicate dishes. Open circles 10(O) show 1a,25(OH)ZD3~ closed circles (~) show analog HS.
In terms of the results presented in Figure 10, it is clear that analog HS is significantly more potent than loc, 25 (OH) 2D3 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 la, 25 (OH) 2D3 and is theref ore about twenty times more potent at promoting the cell differentiation of HL-60 cells.
vI. Ra~$,~syonses Rapid responses are initiated by occupancy of the VDR~", with an analog ligand that has the shape of a 5-s-cps oriented 1a,25(OH)2D3. Rapid responses of the analogs of the invention are detected by their ability to achieve transcaltachia or mitogen activated protein kinase.
A. 'r'ranscattachia 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 [ia,25(OH)ZD3~ or, according to the current invention, by 6-s-cps conformationally restricted analogs. The transcaltachia is a rapid response which occurs within one to 3o 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-cps conformationally restricted analogs are described below.
Transcaltachia is a component of the overall process describing the intestinal absorption of calcium, which is the SUBSTITUTE SHEET (RULE 28) classic response related to the vitamin D. For the intestinal absorption of calcium in humans 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 D3 that is responsible for the stimulation of intestinal calcium absorption is a vitamin D metabolite loc, 25 (OH) ZD3, 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 ad j scent 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-cis shape or by 6-s-cis locked analogs of the invention which are delivered by vitamin D binding protein (DBP) to the exterior surface of the epithelial cell. There, the hormone D
or the 6-s-cps locked analog is unloaded from the DHP in its free form immediately adjacent to the outer cell membrane of an epithelial cell where the receptor VDR"~" is resident, as shown in Figure 4. The vD~", is 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"~/6-s-cis analog, results in the generation of a biological signal involving opening of voltage-gated calcium channels that send a massage to the interior of the cell so that there is a prompt (rapid) initiation of the export of the calcium bearing lysosomal-like vesicles. Hence this activity is identified as a rapid response. This export process occurs within 1-3 SUBSTITUTE SHEET (RULE 26) minutes. Thus, the net effect of the delivery of a 6-s-cis 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 5 exiting of calcium from the interior of the epithelial cell into the blood compartment. Thus, the process of transcaltachia increases the availability of calcium for delivery to the bone system where it is utilized for an increase in bone mineral content and density.
10 Figure 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 confonaationally flexible 1a,25(OH)ZD3, which is able to achieve the shape of the 15 6-s-cis locked conformationally restricted analogs and thus interact with the VDR,a~", which has been implicated in transcaltachia.
Findings that only 6-s-cis locked analogs can elicit transcaltachia is extremely important for their therapeutic 20 utility. While 1a,25(OH)2D3 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 25 danger of causing hypercalcemia which can happen if large doses of 1a,25(OH)2D3 are administered. Such doses inappropriately activate the bone resorbing cells or osteoclasts.
Figure 11 represents stimulated °SCa2' transport in duodenal loops vascularly perfused with 1a,25(OH)ZD3 or 30 1a,25(OH)2-?-dehydrocholesterol (JM), or 1a,25(OH)2-lumisterol (JN). Duodenal loops from normal, vitamin D-replete chicks were lumenally perfused with 'SCa2+ (5 uCi/ml of buffer) . To establish basal transport rates, celiac artery of controls were perfused with control medium for the first 20 min. The duodena were then 35 either re-exposed to control medium containing the vehicle ethanol (0.005%, final concentration) through the celiac artery, SUBSTITUTE SHEET (RULE 28) WO 99116452 PCT/LJS981198b2 or vascularly perfused with 300 pM or 650 pM agonist analogs JM
or JN or with 650 pM of a control reference compound la, 25 (OH) 2D,. The venous effluent was collected at 2 min intervals for liquid scintillation spectrophotometry of the '5Ca2+. The results obtained during the treated phase were normalized to the average basal transport for each duodenum.
Values represent mean ~ SEM for n = 4 in each group.
Figure ilA shows results obtained after perfusion with analog JM. Figure 11B shows results obtained after perfusion with analog JN. Included in each graph are both the vehicle control and 650 pM 1a,25(OH)2D, of reference compound as a positive control. The results seen in Figures 11A and 118 indicate that the 6-s-cis locked analogs JM and JN are potent analogs of the rapid response process of transcaltachia. As seen in these figures, within first four minutes, both analogs have activity comparable or better than the reference compound.
As also seen in Table il, analog JM has 60% of the potency of the conformationally flexible 1a,25(OH)ZD, to stimulate transcaltachia, while analog JN is 105% as potent as 1a,25(OH)2D3. Additionally, a 6-s-trans conformationally locked analog JH [1(,25(OH)-tachysterol,] was found to have smaller than 5% activity of 1a,25(OH)2D3 in stimulating transcaltachia.
From these results it is clear that only the 6-s-cis conformational analogs are the active agonists for rapid responses.
g, ~ti_tor~en Activated Protein IC,'_r~ase 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 mitogens 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.
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 SUBSTITUTE SHEET (RULE 26) involved in modification of genomic responses of cells, for example, including their differentiation and/or proliferation.
1a,25-dihydroxyvitamin D3 and particularly its 6-s-cis analogs are selective agonists of cytosolic localized mitogen activated protein (MAP)-kinases. Further, 1a,25 dihydroxyvitamin D3 (analog HL) is an antagonist of activation of MAP-kinases. These findings may be advantageously used in a method for activation or inhibition of vitamin D-related rapid responses. The method of the invention is useful for selective and rapid treatment of various diseases in which drug forms of vitamin D3 and its metabolites are involved.
It has now been additionally discovered that the analogs of 1x,25-dihydroxyvitamin D3 mediate activation of MAP-kinases, particularly MAP-kinase p42°'°p'' phosphorylation, in a time and dose-dependent manner.
For the purposes of this study, three 6-s-cis locked analogs, namely HF {1a,25(OH)2-previtamin-D3, JM {1a,25(OH)2-7-dehydrocholesterol), and JN (1a,25(OH)2-lumisterol3) and one 6-s-trans locked analog, namely JB (1a,25(OH)-tachysterol3) were prepared and studied for their ability to rapidly activate the MAP-kinase p42"'pk pathway.
Such activation was achieved and mediated only by 1a,25(OH)ZD3 analogs which can assume conformation that is closely approximated by the 6-s-cis conformation of 1a,25-dihydroxy-7-dehydrocholesterol and 1a,25-dihydroxylumisterol.
In order to determine whether MAP-kinase phosphorylation is specific and is altered by 1a,25{OH)2D3, the time-dependent effects of 1a, 25 (OH) 2D3 on p42m°p'' phosphorylation was examined using human acute promyelocytic leukemia cells {NB4). In this 3o study, the N84 cells, cultured in 10% charcoal-stripped fetal calf serum (FCS) medium, were treated with la, 25 (OH) ZD3 at 10-8M
for various time periods. Cells were then extracted and the phosphorylated MAP-kinase was immunoprecipitated with anti-phosphotyrosine antibody and further analyzed by Western blot using the antibodies against p42°°p''.
Specificity of p42°'p'' phosphorylation by la, 25 (OH) ZDj in N84 SUBSTITUTE SHEET (RULE 28) cells is shown in Figure 12. Figures 12A and 12B present the results of a densitometric scan of the Western blot analysis.
For studies illustrated in Figure 12, the NB4 cells were treated with 1a,25(OH)ZD3 at 10'eM for 5 min and then extracted as described in Example 7. The lysate was further processed for anti-phosphotyrosine immunoprecipitation. 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°'°p'' antibodies that were (+) or were not (-) pre-exposed to MAP-kinase peptide.
Figure 12A shows results of a dose response by 1a,25(OH)2D3 for activation of MAP-kinase at either 1 or 5 minutes exposure to it. As seen in Figure 12A, 1a,25(OH)2D3 significantly increased phosphorylation of p42"'ap'' 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 1a,25(OH)ZD, and a 6-s-cis 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 1a,25(OH)2D3 and were only slightly less active at 5 minute intervals.
VII . Antagonist Anal oars A. Genomic An~agon~~
Genomic antagonists are compounds that function as antagonists of the vitamin D nuclear receptor. The genomic antagonists are believed to cause the VDR""~ to assume a conformation which blocks transcriptional machinery.
B. Nonaenomi c-Ra~m~nonse An~g~n; ~sta Rapid response antagonists are compounds that function to antagonize the DVR,~~. One representative conformationally flexible genomic antagonist is analog HL, namely 18,25(OH)2D3.
Figure 13 illustrates the ability of iB,25(OH)ZD3 to inhibit SUBSTITUTE SHEET (RUL.E 26) the agonist actions of 1a,25(OH)ZDj on the rapid response of transcaltachia.
For this study, the 18,25(OH)2D3 analog HL was added to the perfused duodenum either in advance or simultaneously with 1a,25(OH)2D3 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 1a,25(OH)2D3. Open circles represent the negative control receiving no treatment with 1a,25(OH)2D3 or analog. Figure 13B shows the dose-response relationship of 1B,25(OH)2D3 inhibiting the stimulation of transcaltachia by 300 pM 1a,25(OH)2D3. Data represent the ratio of treated to basal values ~ SEM extracted from a time-course plot (as in panel A) at 32 minutes.
The transcaltachia caused by 1a,25(OH)ZD3 was particularly observable in Figure 13A-1 where the antagonist HL was tested at 12 pM in combination with 1a,25(OH)ZD3 at 300 pM. When the antagonist was added at 60pM in advance of 30opM 1a,25(OH)ZD3 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 1a,25(OH)2Dj.
When the antagonist was added at 400 pM and the 1a,25(OH)ZD3 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 1a,25(OH)2D3, transcaltachia was almost completely inhibited and the transport of the calcium ion across the intestinal wall was inhibited.
The results presented in Figure 13 document the potent ability of 18,25(OH)2D3 (HL) to block or antagonize the action of the conformationally flexible la, 25 (OH) ZD3 to stimulate the rapid response of transcaltachia. These results further show that the antagonist analogs of the invention are able to inhibit the agonist activity of the native hormone D as well as that of agonist analogs of the invention.
Utility of iB,25(OH)2D3 and other antagonist is based on their ability to inhibit the normal rapid actions of 1a,25(OH)ZD3 or other agonist and to block the intestinal absorption of SUBSTITUTE SHEET (RULE 26~

calcium when the individual has an abnormally elevated blood concentration of Ca2+ in blood. Antagonists of the invention are, therefore, useful for treatment of conditions such as hypercalcemia. They prevent exacerbation of the extant 5 condition of hypercalcemia.
In other experiments the analog iB,25(OH)2Dj (HL) has also been found to be capable of antagonizing rapid responses of 1a,25(OH)ZD3 to stimulate the opening of chloride channels in ROS
17/2.8 cells in osteoblast cells and the activation of MAP
10 kinase in human leukemia cells.
Analog's HL antagonist action is illustrated by its ability to inhibit the rapid responses of 1a,25(OH)ZD3. These antagonist actions are illustrated in Figures 14 and 15.
Figure 14 shows opening or modulation of chloride channels 15 in osteoblastic ROS 17/2.8 cells, following stimulation by 1a,25(OH)ZD3. Specifically, Figure 4 shows fold increase of outward currents in ROS 17/2.8 cells mediated by 1a,25(OH)2Dj in the absence and presence of 1 nM 1ø,25(OH)2D3. Fold increase of current amplitudes promoted by different concentrations of 20 1a,25(OH)ZD, were measured for currents elicited by a depolarizing step to 80 mV, in the absence and presence of 1 nM
HL in the bath. In each case, at least a 3-min period was allowed after the addition of the analog to the bath for currents to reach a stable amplitude value. Currents were 25 obtained in the presence of glutamate as the permeant anion since seals were more stable and long lasting than in the presence of C1-. Anion currents were isolated from inward Ba2+
currents after blockade of Ca2+ channels with 100 ~cM Cd2+.
1a,25(OH)2D3 alone showed a concentration-dependent effect on the 30 promotion of anion currents (14 out of 15 cells, 93~), with a maximal value obtained for 0.5-5 nM hormone (black bars). In the presence of 1 nM 1ø,25(OH)ZD3 (white bars), the potentiation effect by la, 25 (OH) 2D3 was significantly reduced ( *, p < 0. 05;
**, p < 0.01, n = 3-8) for a concentration of the hormone of 5 35 nM or less.
As seen in Figure 14, the synthetic analog 1ø,25(OH)2D3 (HL) SUBSTITUTE SHEET (RULE 26) WO 99116452 PC'TlUS98/19862 which only differs from a natural metabolite in the orientation of the hydroxy group on carbon 1, has been shown to inhibit the ability of 1a,25(OH)2D3 to increase outward currents, that is, to open chloride channels in ROS 17/2.8 cells. Thus, 1a,25(OH)2D3 acting alone, over the range of 0.05-50 nM, is an agonist which opens chloride channels, but the addition of 1Q,25(OH)2D3 at 1 nm blocks this agonist actions of 1a,25(OH)2D3, Figure 15 illustrates the stimulation of activation of MAP
kinase, specifically stimulation of phosphorylation of MAP
lo kinase by 1a,25-dihydroxyvitamin D, in promyelocytic NB4 leukemia cells.
Figure 15 shows the effect of analog HL on 1a,25(OH)ZD3-induced p42"'~Pk phosphorylation in NB4 cells. (A) NB4 cells were treated with different doses of 1a,25(OH}2D3 in the presence or absence of HL at 10-9 M for 5 min. (B) Equal loading of total MAP-kinase proteins was shown. (C) Quantitation of band density of the activated MAP-kinase is expressed as percent of control (set to 100%} from three separate experiments and is shown as the mean ~ SEM. *, P<0.05 compared the HL-treated group with non HL-treated group.
As shown in Figure 15B and 15C, ip,25(OH)2D3 {analog HL) present at a concentration of 10'9 mol was able to block 1a,25(OH)2D3, present at either 1, 10 or 100 x 10-1° M, mediated activation of MAP-kinase. As seen in Figure 158 and 15C, when analog HL was present alone, there was no stimulation of MAP-kinase.
These results clearly show the antagonistic effect of analog HL on the rapid responses generated by 1a,25(OH)2D3.
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.
SUBSTITUTE SHEET (RULE 28) VIII . Theraneutt c Uti t i t-yr of The nr~.lo~w of the Invents ng A. Eya_1_uati_on of Therap »t-ie~ Ut 1 sty of the Analycac, From the perspective of drug development relative to analogs of 1a,25(OH)2D3, the primary objective is to identify an analog which has activity similar to or better than hormone D
but which has more specifically defined properties with respect to binding to nuclear or membrane receptors but which does not lead to hypercalcemia. The ideal analog of 1a,25(OH)ZD, should have a much lower intrinsic ability to elevate the blood concentration of calcium than the parent 1a,25(OH)2D3 hormone.
Analog's profile evaluation includes as the first step, its evaluation of its ability to interact with the VDR~",~ and DBP
binding proteins under in vitro steroid competition assays, as outlined in Figures 7 and 8. Next, a given analog's ability to stimulate intestinal Ca2' absorption (ICA) and bane Ca2 mobilizing activity (BCM) in the vitamin D-deficient chick bioassay is screened. This determines the potency of the ICA
and BCM calcemic responses that the analog can generate is vivo over a 24 hour interval. Positive results of these assays indicate analog utility as a drug of choice for disease where the calcium absorption is disturbed, such as osteoporosis, rickets, etc. Next, the analog is screened to determine its relative ability to mediate classic genomic responses and/or rapid responses in a whole cell or in vivo setting. The classic genomic responses are determined using tissue culture conditions for the analog cell differentiating ability, as seen in Figure 10, while the rapid responses are tested in assays that allow quantitation of MAP-kinase activation in NB4 cells and elicitation of transcaltachia. Results obtained in these assays delineate the analog as the drug of choice for treatment of acute hypocalcemia or chronically present hypocalcemic syndrom.
Additionally, when the analog is found, for example, to be inhibitory in a cell proliferation assay, it becomes a good candidate for treatment of cancer growth or leukemia.
Then, depending upon the nature of the analog under study that is depending whether or not the analog is conformationally SUBSTITUTE SHEET (RUL,E 26) flexible (e. g., analogs EV, JV, LO), conformationally restricted (e. g., analogs JM, JN), or an antagonist of rapid responses (e.g., analog HL; see Table 1), 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. At the same time, the toxicology of in vivo chronic dosing with respect to the hypercaicemia-toxicity assay listed in the bottom line of Table 7, is performed and the analog is evaluated for its potential therapeutic activity.
g. Animal Models of Human Disease States In order to extrapolate the results obtained in cell culture and to identify and evaluate new analogs of 1a,25(OH)2D3 which possess favorable therapeutic attributes in a variety of human disease states, it is essential to have access to appropriate animal is vivo model systems. Such model systems allow a critical evaluation of new drugs, in this case, of the analogs of the invention for the mediation of favorable responses, as well as allowing detection of the onset of unfavorable or toxic responses.
Table 12 presents a summary of animal models that have shown a demonstrated utility for drug development studies in the vitamin D endocrine system.
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1. , 39 Suppl. 1:18-23, (1990).

2. ~"~, 39 Suppl. 1:24-26, (1990) .

3. Transt~l. Immunol,s,, 1:72-76, (1993) .

4. Vitamin D. Molecut_ar_ Cellular and Clfnical , pp. 346-347, Berlin, New York: Walter de Gruyter ( 1998 ) .

5. ~, at 334-335.

6. Transplant. Proc., 26: 3128-3129, (1994).

7. , 37: 552-558 (1994).

8. Cl,'_n. Ext~. hnmunol,, gg; 301-306 (1992).

9. J. Clin. Invest., 87: 1103-1107, (1991).

10. C in. Immuno Immunoy, 54: 53-63 (1990).

11. J. Cell. Biochem., 49: 26-31 (1992).

12. J. Nutr. Sci. V'taminol_'~ 31: S44-S571985.

13. Am. Rev. Res~,i_r. Dis., 138: 984-989 (1988).

14. Exn. I~Iematol,.,,., 13: 722-732 (1985) .

15. Endocrinoloav, 129: 832-837 (1991).

16. Anticancer Drucs, 2: 475-480 (1991).

17. Cancer Lett., 55: 149-152 (1990).

18. J. Endo., 141: 411-415 (1994).

19. Brit. J. Dermatol_, 132: 841-852 (1995).

20. Acta Dean. Venereol . Str,racl, _ t ~, 77: 196-202 (1997).

21. Urn, 50: 999-1006 (1997).

22. Vitamin D: Biochemical,, Chemical_ and Clinical As;a Related to Calc;_um Met~~l~~ pp, 587-589, Berlin:

Walter de Gruyter (1977).

B, A_n_a_1_o~ Del i yery to the Tiss ~ps~/ ~arø
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. The key role played by the vitamin D-binding protein (DBP) in the transport of 1a,25(OH)2D3 or its analogs through the blood compartment, from its site of production or uptake, to make them available for uptake by target cells in tissue or organs to be treated has bean illustrated in Figure 4.
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 1a,25(OH)2D3, 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 SUBSTITUTE SHEET (RUL.E 28) 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""~ or the VDF~"n, and assert its therapeutic potential.
C. TherapeLti c Effect of la,. 25 (OH1 ~~ on Snecif i c Vitami r D Diseases - Clinical Applications A. Ar~~onist naloas The agonist analogs of the invention are useful for treatment or prevention of various diseases caused by or accompanying the deficiency or overproduction of vitamin D, particularly a deficiency of its metabolite 1a,25(OH)ZD3. For treatment and/or prevention of these diseases, pharmaceutical 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 14a,15a-methano-1a,25(OH)2D3 (LO), 22 (m(dimethylhydroxymethyl)phenyl-23,14,15,16,17-pentanor ia(OH)D3 (EV), or 1a,18,25(OH)3D3 (HS). 6-s-cis locked analogs of 1a,25(OH)2D3 subject to this invention which are listed above in Table 4 are exemplarized by analog 1a,25(OH)2-lumisterol (JN) These 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 3o these diseases are caused by the vitamin D or its metabolites deficiency or may be corrected by treatment with vitamin D
metabolites, particularly 1a,25(OH)2D3.
Treatment and Preyent i an o Ost'pnt~nrnc i c 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 SUBSTITUTE SHEET (RULE 26) 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.
The rationale for utilization of analogs of 1a,25(OH)2D3 in the treatment of osteoporosis is based on the documented decrease in serum concentrations of 1a,25(OH)2D, in elderly subjects. When the serum level of 1a,25(OH)2D3 decreases, the calcium intestinal absorption is impaired. Administration of supplementary 1a,25(OH)ZD3, or an analog equivalent thereof, corrects this conditions and results in improvement of the calcium absorption from the gut. That, in turn, leads to increased availability of calcium for bone structure and in increased mineral bone content and increased bone density. 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 1a,25(OH)ZD3 and for treatment and prevention of osteoporosis.
Particularly active for treatment of osteoporosis are the drug formulations of the 1a, 25 (OH) ZD,, such as the conformationally flexible analogs LO [14,15-methano-1a,25(OH)ZD3], EV [22-(m(dimethylhydroxymethyl)phenyl-23,14,15,16,17-pentanor la(OH)D3], or HS [1a,18,25(OH)3D3] or the drug formulations of 6-s-cis locked analogs of 1a, 25 (OH) 2D3, such as analog JN [1a,25(OH)Z-lumisterol]. These drugs are used to treat those forms of osteoporosis which are related to a lowered level of serum 1a,25(OH)2D3, because they rapidly stimulate intestinal Ca2+ absorption thereby increasing the fraction of the dietary Ca2+ that is absorbed by the intestine and made available to the skeletal system. In addition, 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 1a,25(OH)2D3/70 kg body weight, taken daily. The treatment duration is continuous for SUBSTITUTE SHEET (RULE 2fi) treatment of elderly patients and those with documented osteoporosis with serum Ca2+ 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.
Treatment of osteoporosis is exemplarized in Example 8.
Trpa_tment and Prevention of Osteomalacia and Rickets 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, whereas 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.
One of the primary causes of osteomalacia and rickets are disorders in vitamin D endocrine system. Such a problem may be increased due to insufficient sunlight exposure, nutritional vitamin D deficiency, the nephrotic syndrome and malabsorption or abnormal metabolism of vitamin D. Two types of vitamin D
dependent rickets are known.
Vitamin D-dependent rickets type I is a recessive disease in which there is a low level of 1, 25 (OH) ZD resulting from a selective deficiency in the renal production. To treat this condition, moderate doses of vitamin D (0.625 ~cg) or physiological doses (0.5-1 microgram) of 1,25(OH)ZD3 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)2D,however, due to a mutation in the vitamin D receptor which reduces the affinity of the receptor for its SUBSTITUTE SHEET (RULE 26) ligand 1,25(OH)ZD and therefore it does not function properly.
To treat this condition, large doses of 1,25(OH)2D3 (20-60 micrograms) are used.
Adults with osteomalacia or children with rickets have a blood Ca2' concentration significantly below the normal range of 9.0-10.5 mg/100 ml. The serum Caz+ concentration in the disease state may be as low as 5.0-8.0 mg/100 ml. In addition, afflicted individuals typically have high levels of serum alkaline phosphatase, a marker for bone disease.
To treat adult osteomalacia, any of the drug formulations of the 1a,25(OH)ZD3 conformationally flexible analogs which during testing were able to elicit both the rapid responses and genomic responses are suitable for treatment of osteomalacia.
Thus, the 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. Similarly, also suitable are formulations comprising 6-s-cis locked analogs JM, JN, JO and JP.
These drugs cause increase in the dietary Ca2+ absorption by the intestine by promoting transcaltachia and by making calcium and phosphate available to the skeletal system to assure adequate mineralization of bone. By providing the substitute analogs of the vitamin D, the osteoblast is activated and begins to produce bone matrix that can be mineralized.
The analog of the la, 25 (OH) ZD3 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 1a,25(OH)2D3/70 kg body weight, daily. The dose is appropriately modified for children.
The treatment duration depends on the treated conditions.
For treatment of vitamin D-dependent rickets type I, the child is treated until the bone mineralization is normalized.
This is likely to take several months or even years. Example 9 illustrates the treatment regimen. For treatment of rickets type II, the child is treated with larger dosages of the analog and, its serum Ca2+ levels are monitored weekly until the SUBSTITUTE SHEET (RULE 26) appropriate level is detenained. 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.
5 Treatment of adult osteomalacia is achieved in the same manner as described for treatment of osteoporosis.
T_rpatment and Preventio_n_ of Renal Osteodystrotihv Renal osteodystrophy is a bone disease that occurs in association with chronic renal failure. Chronic renal failure 10 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. Over time of continued use of the dialysis procedure, however, renal osteodystrophy 15 develops because the normal endocrine function of the kidney is compromised resulting in an impairment of the 25(OH)D3-1-hydroxylase synthesis. This hydroxylase is responsible for the enzymatic production of the steroid hormone, 1a,25(OH)ZD,.
Accordingly, patients suffering from chronic renal failure 20 inevitably become hormone D [1a,25(OH)2D3] deficient. As a consequence, 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). The PTH's secondary action in the 25 instance of hypocalcemia is to stimulate the bone resorbing cells (osteoblasts) to mobilize bone calcium and make it available to the blood Ca2+ pool.
Patients who are diagnosed with renal osteodystrophy display a reduced serum level of 1a,25(OH)2D;, a reduced level 30 of intestinal Ca2+ absorption, increased level of secretion of PTH and a greatly increased level of bone Ca2+ mobilizing activity as stimulated by the excess PTH. In addition, the serum level of Ca2' is reduced to levels 7.5-9.0 mg Ca2+/100 ml.
The main components of renal osteodystrophy are osteitis 35 fibrosa and osteomalacia. Osteitis fibrosa is a pathological condition which develops as a consequence of an increased level SUBSTITUTE SHEET (RULE 26) 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 above. Intestinal absorption of calcium is reduced. Low levels of 1,25(OH)2D3 in serum are observed. Not only these low levels of 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 the inhibitory effect of 1,25(OH)ZD3 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 conformationally flexible analogs or 6-s-cis locked analogs of 1a,25(OH)ZD3, are effective in stimulating the increase of intestinal Ca2+ absorption and thus preventing a detrimental effect of parathyroid hormone leading to renal osteodystrophy.
In addition, these analogs act on the osteoblast cells via 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 serum ionized Ca2+ levels and the secretion of PTH. The parathyroid gland possess both VDR""~ and VDR",~" which participate in the processes governing the secretion of PTH.
For treatment and prevention of renal osteodystrophy, the analog is formulated to achieve in oral dosage an equivalent of 0.5-2.0 micrograms of ia,25(OH)ZD3/70 kg body weight taken daily.
The treatment is continued as long as necessary. Serum Ca2+
levels, alkaline phosphatase levels and the serum level of immunoreactive PTH is monitored every two weeks until stabilization of conditions and then on a monthly basis. The bone mineral density is determined at least once monthly.
SUBSTITUTE SHEET (RULE 28) TrraatlnETl~ pf P~gj,g 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""~] and membrane [VDR~"] receptors for 1a,25(OH)2D3. Under cell culture conditions, keratinocytes have been shown to display both genomic and rapid responses to 1a,25(OH)2D3 and related analogs.
The action of the vitamin D hormone (1a,25(OH)2D3) 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 ~i (TGFp), 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.
icx,25(OH)ZD3 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.
Two types of formulations are used. An analog is formulated for oral administration to achieve an oral dose equivalent to 0.5-2.0 micrograms of 1a,25(OH)2Dj/70 kg body SUBSTITUTE SHEET (RULE 28) 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~cg/gram) of the drug formulations of the la, 25 (OH) 2D3 conformationally flexible analogs or topical formulations of 6-s-cis locked analogs of 1a,25(OH)2D3, are used to treat individuals with external plaques of psoriasis.
Treatmeni~ and Prevention of Leukemia 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 1a,25(OH)2D3 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.
1a,25(OH)2D3 is known to be an effective inhibitor of human leukemia cell proliferation and as well a stimulator of the cell differentiation. There have been a wide array of studies utilizing analogs of 1a,25(OH)ZD3 on human leukemia cells in tissue culture as described in Blood, 74: 82-93 (1989). In addition, animal models for study of leukemia treatment are available as outlined in Table 7.
Human leukemia N84 cells have been shown to_have both VDR""
and VDRm~" and display both genomic and rapid responses to 1a,25(OH)2D3 and its analogs.
The drug formulation of the analog is oral or IV, containing 1-10 micrograms per day. In the initial treatment stage, the higher doses of the analog are administered intravenously or intraperitoneally. Treatment typically lasts 7-21 days but may last as long as necessary. The endpoints of SUBSTITUTE SHEET (RULE 26) 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.
Inhibit; on of Groa,~h of Prostate Cancer Cells Prostate cancer is the most common non-skin cancer among men in many Western societies. Nearly 50% of all prostate l0 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 1a,25(OH)ZD3 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""~and VDT.
Because of their antiproliferative activity, 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 ,ug 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 Util,'_ty For Organ Trans,~,lantatinn The vitamin D endocrine system includes the immune system in its sphere of actions. Both activated T and B lymphocytes have the VDRt",~ and VDR"~". Although the physiological role of 1a,25(OH)2D3 in the immune system is not yet clearly defined, 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 SUBSTITUTE SHEET (RULE 26) la, 25 (OH) 2D3 .
Importantly, the natural killer cell activity is also enhanced by 1a,25(OH)aD,. This enhancing effect of the nonspecific immune defense contrasts with an inhibition of the 5 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 la, 25 (OH) 2D,.
As summarized in Table 7 several animal models have been used to evaluate the effect of 1a,25(OH)2D3 and its analogs on organ 10 transplantation and resection. These results support utilizing analogs of 1a,25(OH)ZD, to counter immunoreactions connected with human organ transplantation, such as kidney transplantation, heart, or combined heart and lung transplantation, skin transplantation, and pancreas transplantation.
15 Therat~eutic Action of Antagonist Analogs The analog HL [18,25(OH)2D3] which is an antagonist for the rapid actions mediated by 1a,25(OH)2D3 is suitable to treat individuals experiencing hypercalcemia, particularly individuals with elevated plasma levels of 1a,25(OH)2D3 occurring 20 in primary hyperparathyroidism or drug overdose of 1a,25(OH)2D3 or la,(OH)D3 with drugs Rocaltrol or Alpherol.
The clinical hypercalcemia describes circumstances where the blood concentration of Caz+ is elevated above the normal range of 9 . 0-10. 5 mg Ca2+/ 100 serum. Elevations of blood Ca2+
25 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 Ca2+ levels above 12.0-13.0 mg/100 ml are frequently treated by hemodialysis with a low concentration of Ca2' in the 30 dialysis bath in an effort to acutely lower their prevailing serum concentration of Ca2' to the normal range.
If, however, the causative factors which produced the hypercalcemia, e.g. primary hyperthyroidism or 1a,25(OH)ZD3 intoxication, are ongoing, the excess levels of 1a,25(OH)2D, 35 inappropriately stimulates intestinal Ca2+ absorption and bone Ca2+ mobilizing activity. This process results in additional SUBSTITUTE SHEET (RULE 28j Ca2+ being made available to the blood compartment from both the intestine dietary Ca2+ and bone calcium (hydroxyapatite mineral), which is likely to result in hypercalcemia.
Treatment of the conditions with analog HL (1B,25(OH)ZD3) which is a known antagonist of the rapid responses of transcaltachia, that is, it inhibits the intestinal Ca2+
absorption and also the opening of Ca2+ channels in osteoblast cells and thereby inhibiting bone Ca2+ resorption by nearby osteoclasts.
Hypercalcemic patients are treated with oral or intravenous formulations of 18, 25 (OH) 2D3, 10-50 micrograms every 12 hours.
The effectiveness of treatment is determined by lowering and the absence of a further increase in the serum Ca2+ level, and its fall to a more normal value.
IX. Pha_rmaceLtical_ Comnosi_tions and Administration 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.
The 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. In addition to the analog of the invention or the pharmaceutically acceptable salt thereof, the 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 1a,25(OH)2D3.
SUBSTITUTE SHEET (RULE 28) 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 10%/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. However, these amounts may differ, depending of the intended use and the composition may, in some instances be formulated as the analog without any excipient.
For solid compositions of the analog of the invention particularly suitable for oral administration, conventional non . toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
For oral administration, a pharmaceutically acceptable non-toxic 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 2o suspensions, such as tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain 0.1%-95% of active ingredient, preferably 1%-70%.
When the analog is formulated as suppositories for systemic administration, traditional binders and carriers include for example polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing active ingredient in the range of 0.5%-l0%, preferably 1-2%.
Liquid pharmaceutically administrable compositions suitable for oral or parenteral administration can, for example, be 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. The carrier may optionally contain pharmaceutical adjuvants. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting SUBSTITUTE SHEET (RULE 26) or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate , triethanolamine of eate , etc . P a r a n t a r a 1 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.
In addition, if desired, the pharmaceutical 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.
Actual methods of preparing such compositions and dosage forms are known, or will be apparent, to those skilled in this art. For example of preparing compositions of the invention, see Rem nq~ton ~ s Pha~-~n_a_ce ~t; ~a 1 ~~,~,nces, Mack Publishing Company, Easton, Pennsylvania, 15t'' Edition, 1975. The composition or formulation to be administered will, in any event, contain a quantity of the analogs) 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.
Administration of an active compound, that is the analog of the invention, alone, in admixture or in combination with other compounds, in a pharmaceutical composition 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. Depending on the intended mode of administration, the composition may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, SUBSTITUTE SHEET (RULE 26) WO 99!16452 PG"fIUS98/19862 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 ~Cg/kg/day, preferably 0.01-3 ~cg/kg/day. For an average 70 kg human, this would amount to 0.07-1000 ~g per day, or preferably 0.7-210 ~cg/day.
T1TT~T.TTV~
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 D3.
They are therefore useful for treatment and prevention of diseases connected with either insufficiency or with overproduction of 1 a,25- dihydrvxy vitamin D3.
ExBM~
The following examples describe preparation of specific analogs. Schemes A-J illustrate preparation of the analogs as indicated.
Scheme A shows synthesis of analogs DE, DF and EV
described in Examples 1-3.
SUBSTITUTE SHEET (RULE 28~

R
R
5 I!I i I H 2abc O T f ~ ~ I H T 30MS~
T30MSC OTBDbIS 3a,b,C p~ H.,. P~, ljndlar PdlPPh Ci III qumcune. nexanas ~)2~ 12.
Cul, e! ~~H, dhlF ~ i H
~ ~ ~ T 30htS0 4a,b,c lO R
) 3~ Isocc:ane.
retlux 4) ToaF, H THF
Sa,b,c (DE, DF,EV respectively) HO' OOH
wherein the R group of a, b or c is;
- ~~~oEt (oT~s~
~~~OH (OTHDbtS) '~~OH (OTBDMS) I
a b c In compounds 2, 3 and 4, the side chain is protected as the silyl ether; in compound 5, it is the free OH.
FxA~r,E i Ohem,'_cal Synthes,'_s of Analog D
This example describes preparation of analog DE, namely 22-(m-hydroxyphenyl)-23,24,25,26,27-pentanor-la(OH)D~
according to Scheme A.
1a,3~i-Di-(tert-butyldimethylsilyloxy)-22-(m-tert butyldimethylsilyloxy)phenyl-24-nor-9,10-seco-chola-5(10),8 dien-6-yne, compound (3a).
The A-ring fragment 1 (0.077 g, 0.14 mmol) and 0.060 g SUBSTITUTE SHEET (RULE 26) (0.16 mmol) of the CD ring triflate (2a) were dissolved in 0.6 mL _of dry DMF. Bis-triphenylphosphine palladium dichloride complex (Pd(PPh3)C12, 3 mg) and diethylamine (0.076 mL, 0.55 mmol) were then introduced. The mixture was heated to 80°C
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%
HC1, a solution saturated of NaHC03 and brine. After drying (MgS04) and concentrating the solution, the crude residue was 1o passed through a short column of silica gel (1% EtOAc/hexanes) and then purified by HPLC (Rainin Dynamax-60A column, 0.4%
EtOAc/hexanes, 8 mL/min) to afford 86 mg (81%) of the dienyne 3a as a colorless residue.
Spectral Data: l~: 5 0.09 (6H, Me2Si, s), 0.12 (6H, Me2Si, s) , 0.21 (6H, MezSi, s) , 0.76 (3H, C18-Me, s) , 0.86 (3H, C21-Me, d, J~6.3 Hz), 0.91 (9H, t-Bu, s), 0.92 (9H, t-Bu, s), 1.00 (9H, t-Bu, s} , 1.93 (3H, C19-Me, s) , 2.43 (1H, dd, J"'3.6 Hz, 16.2 Hz), 2.87 (1H, dd, J-2.1 Hz, 13.2 Hz), 4.12 (1H, Hl, m) , 4.22 (1H, Hl, m) , 6.00 (1H, H9, m) , 6. 64 (1H, ArH2, s) , 6.68 (1H, Ar, d, J-8.4 Hz), 6.76 (iH, Ar, d, J'7~5. Hz}, 7.13 (1H, ArHS, t, J"7.8 Hz).
Cue:
13 S -4.5, -4.4, -4.3, -4.1, -4.0, 11.3, 18.3, 18.4, 18.6, 19.4, 24.5, 25.4, 26.0, 26.1, 26.2, 28.9, 36.1, 39.1, 40.1, 41.5, 42.2, 42.8, 50.5, 55.3, 64.4, 70.3, 88.4, 92.6, 115.7, 117.5, 121.5, 122.6, 122.7, 129.1, 133.5, 140.7, 143.3, 155.6.
m/z 762.5303 (calcd. for C4sH~803Si3, 762.5259) .
m/z 762 (2, M), 623 (25), 631 (57), 630 (base), 628 (11), 574 (10), 499 (18}, 498 (41), 441 (6), 407 (2), 381 (2), 355 (2), 324 (19), 277 (11), 268 (10}, 249 (11}, 222 (32), 193 (4) , 165 (4) , 132 (3) , 105 (3) , 75 (52) , 56 (2) .
22-(m-Hydroxyphenyl)-23,24,25,26,27-pentanor-la-hydroxyvitamin D3 (5a) analog DE.
Dienyne, compound 3a (26 mg, 0.034 mmol) in 16 mL of EtOAc, 52 mg of Lindlar catalyst and quinoline (52 ~.L, 0.107 M in hexanes) were stirred for 1 h at room temperature under a positive pressure of hydrogen. The mixture was passed through a pad of diatomaceous earth and then the filtrate was evaporated to dryness. The residue in isooctane (14 mL) was refluxed for 2 h. The solvent was evaporated and to-the residue was added 0.95 mL of THF and 0.23 mL of a solution of tetrabutylammonium fluoride (1 M in THF). After stirring the mixture at room temperature for 12 h, 2 mL of a saturated solution of NaCl was added. The mixture was extracted four times with EtOAc and the combined organic extracts were dried (MgS09) and then concentrated to dryness. After filtration of the residue through a pad of silica geI (EtOAc), HPLC
purification (Rainin Dynamax, 1 x 25 cm, 8 ~cm, 4 mL/min, 100%
EtOAc) to afford 8.3 mg (63%) of the vitamin D, compound 5(a) as a colorless, amorphous solid.
Spectral Data: 1~: b 0.58 (3H, C1g-Me, s), 0.83 (3H, CZ1-Me, d, J'6.3 Hz) , 2.32 (IH, dd, J'6.6 Hz, 13.2 Hz) , 2.61 (iH, dd~, J'1.5 Hz, 13.5 Hz), 2.84 (1H, apparent dt, J'2.1 Hz, 12.9 Hz; this signal most likely consists of two doublets both with J'I2.9 Hz assignable to H9s and probably one of the two HZZ protons) , 4.24 (1H, H3, broad s) , 4.44 (iH, Hl, broad s) , 4.60 (1H, ArOH, broad s) , 5.02 (1H, H19, s) , 5.34 (iH, Hi9, s) , 6.04 (iH, H~, d, J'11.4 Hz) , 6.39 (iH, H6, d, J'll-~-4 Hz) , 6.63 (1H, ArH2, S) , 6.64 (1H, Ar, d, J'7.5 Hz) , 6.71 (1H, Ar, d, J'7.5 HZ) , 7.13 (1H, ArHs, t, J'7.5 HZ) . jjy: (95% EtOH) h",ax 268 nm (E 20, 600) .
422.2839 (calcd. for C2gH38~3. 422.2821} . ~: m/z 422 (10, M) , 404 (base) , 386 (12} , 363 (3) , 349 (2) , 334 (2) , 315 (4) , 297 (6) , 269 (10) , 251 (8} , 227 (6) , 195 (9) , 159 (15) , 155 {12) , 152 (7) , 134 (31) , 107 (85) , 91 (34) , 79 (25} , 67 (16) , 55 (23) .
EXB~EZ<~
Chem,'_ca1_ Synthesis of Analog DF
This example illustrate preparation of analog DF, namely 22-(p-hydroxyphenyl)-23,24,25,26,27-pentanor-la-hydroxyvitamin-D3. The preparation of analog DF seen in Scheme A.
Preparation of 1a,3(3-Di-(tert-butyldimethylsilyloxy)-22-(p-tert- butyldimethylsilyloxy)phenyl-24-nor-9,10-seco-chola-5{IO),8-dien-6-yne, compound(3b).
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 Ba0).
Diethylamine (0.054 mL, 0.39 mmol) and bistriphenylphosphine palladium dichloride (2 mmol, 2 mg, Pd (PPh3) ZC12) were added 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% HCl, a saturated solution of NaHCO3 and then brine. After drying (MgS04) and concentrating, the residue was purified by HPLC (Rainin 10 Dynamax-60A column, 0.4% EtOAc/hexanes, 8 mL/min ) to afford 0.061 g (80%) of the dienyne 3b as a colorless, residual oil.
Spectral Data: 1~: b 0.08 (6H, Me2Si, s), 0.12 (6H, Me2Si, S) , 0.20 (6H, Me2Si, S) , 0.75 (3H, C18-Me, s) , 0.84 (3H, C21-Me, d, J'6.0 Hz), 0.91 (9H, t-Bu, s), 0.92 (9H, t-Bu, s), 0.99 (9H, t-Bu, s), 1.93 (3H, C19-Me, s), 2.43 (1H, dd, J'3.6 Hz, 16.2 HZ), 2.85 (iH, dd, J'2.1 Hz, 13.2 Hz), 4.13 (iH, H3, m) , 4.21 (iH, Hl, broad s) , 5.99 (iH, H9, m) , 6.76 (2H, ArH3,5, d, J'8.4 Hz) , 7.00 (2H, ArH2,6, d, J'8.1 Hz) . 1'CNMR~: b -4.8, -4.7, -4.6, -4.4, -4.3, 11.1, 18.0, 18.2, 19.2, 24.3, 25.2, 25.7, 25.8, 25.9, 28.6, 35.8, 39.1, 39.8, 41.3,-41.8, 42.0, 50.2, 55.1, 64.2, 70.0, 88.2, 92.4, 115.5, 119.6, 122.5, 130.3, 133.3, 134.1, 140.4, 153.5.
m/z 762.5289 (calcd. for C46H~8O3S13, 762.5259) .
m/z 762 (2, M), 632 (18), 631 (43), 630 (78), 574 (6), 500 (11), 499 (30), 498 (73), 441 (3), 409 (2), 277 (8), 249 (8), 222 (22), 221 (base), 195 (2), 165 (19), 132 (6), 105 (3) , 75 (93) , 56 (3) .
Preparation of analog DF 22-(p-hydroxyphenyl) 23,24,25,26,27-pentanor-la-hydroxyvitamin-D3, compound (5b).
A mixture of dienyne 3b (0.019 g, 0.025 mmol) in ethyl acetate (11 mL), quinoline (0.17 M in hexanes, 0.040 mL, 0.42 mmol) and Lindlar's catalyst (0.040 g) was stirred under an atmosphere of hydrogen for 1 h. After filtration of the mixture through a short pad of silica gel and concentration, the crude residue was purified by HPLC (Rainin Dynamax, 1.0 x 25 cm, 8 E.cm silica gel column, 0.4% EtOAc/hexanes) . The inseparable previtamin and vitamin mixture was dissolved in isooctane (7 mL) and heated to reflux for 2 h, following which the solvent was removed. The residue was dissolved in THF
(0.5 mL) and tetrabutylammonium fluoride (1 M in THF, 0:117 mL, 0.117 mmol) was added at room temperature. The solution was stirred at 20°C for 12 h. A saturated solution of NaCI (1 mL) was added and then the mixture was extracted with ethyl acetate (4 x 2 mL). The combined organic extracts were dried (MgS04) and then concentrated to dryness. The crude material, after passage through a short pad of silica gel (EtOAc), was purified by HPLC (Rainin Dynamax 1.0 x 25 cm, 8 ~cm, 100%
EtOAc) to afford the vitamin 5b (3.6 mg, 34%) as an amorphous, white solid.
Spectral Data: 1~: S 0.57 (3H, C1s-Me, s), 0.81 (3H, Czl-Me, d, J'6.6 Hz), 2.33 (1H, dd, J'13.5 Hz, 6.6 Hz), 2.61 (lH, dd, J'13.5 Hz, 2.7 Hz), 2.82 (2H, apparent dd, J'13.5 Hz, 2.4 Hz; this signal most likely consists of overlapping doublets assignable to H9~ and probably one of the H2z protons) , 4.24 (1H, H3, m) , 4.44 (1H, Hl, m) , 5.01 (iH, H19, s) , 5.34 (1H, H19, s) , 6.03 (1H, H7, d, J'12.1 Hz) , 6.38 (1H, H6, d, J'll. i Hz) , 6.74 (2H, ArH3,5, d, J'8. 3 Hz) , 6.99 (2H, ArH2,6, d, J'8.3 Hz) . ' jjy: (abs. EtOH) 1s",aX 266 nm (E 20., 000) .
m/z 422.2824 (calcd, for C28H38O3, 422.2821) .
M~: m/z 422 (19, M), 404 (15), 386 (25), 363 (8), 348 (8) , 320 (3) , 297 {9) , 279 (5) , 241 (6) , 223 (7) , 197 (12) , 157 (16) , 155 (12) , 152 (3) , 134 (32) , 107 (base) , 95 (14) , 81 (13), 71 (14), 57 (15), 55 (26).
Chemical Synthesis of Anal2,a EV
This example illustrates preparation of. the analog Ev, nam ely 22-[3-(1'-Methyl-1'-hydroxyethyl)phenyl]
23,24,25,26,27-pentanor-1a-hydroxyvitamin D3. Preparation of analog EV is seen in Scheme A.
Preparation of 1a;3(3-Di(tert-butyldimethylsilyloxy)-22 [3-(1'-methyl-1'-trimethylsilyloxyethyl)phenyl]-24-nor-9,10 seco-choler-5(10),8-dien-6-yne, compound {3c). 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 (PPh3) 2 (OAc) Z, 1 mg of cuprous iodide and 0. 4 mL of Et2NH. 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 NaHC03 and brine.
5 After drying (MgS09), the solvent was evaporated and the residue was filtered through a pad of silica gel, (1% EtOAc hexanes). The crude dienyne 3c was purified by HPLC (Rainin Dynamax, 1.0 x 25 cm, 8 Vim, 0.5% EtOAc/hexanes, 4 mL/min) to afford 42 mg (93%) of dienyne as a chromatographically 10 homogeneous, colorless oil.
Spectral Data: 1~: S 0.09 (12H, 4MeSi, s), 0.11 (9H, 3MeSi, S) , 0.76 (3H, C18-Me, S) , 0.86 (3H, C21-Me, d, J-6.6 Hz), 0.90 (9H, t-Bu, s), 0.91 (9H, t-Bu, s), 1.59 and 1.60 (3H and 3H, diastereotopic MeZC, two s), 1.93 (3H, C19-Me, 15 s), 2.43 (1H, dd, J"2.7 Hz, 15.9 Hz), 2.93 (1H, dd, J-2.1 Hz, 13 .2 Hz) , 4.11 (iH, H3, broad m) , 4.21 (1H, Hl, br s) , 5.99 (iH, H9, m), 7.01 (1H, Ar, d, J-6.6 Hz}, 7.24 (3H, Ar, m).
Cog is b -4.8, -4.7, -4.6, -4.3, 2.3, 11.1, 14.1, 18.0, 18.1, 18.3, 19.2, 22.7, 24.3, 25.2, 25.8, 25.9, 26.0, 28.7, 20 31.6, 32.3, 32.7, 35.8, 39.0, 39.8, 41.3, 42.0,--42.8, 50.2, 55.1, 64.2, 70.0, 75.2, 88.2, 92.4, 115.5, 121.9, 122.5, 126.0, 127.3, 127.5, 133.3, 140.4, 140.9, 149.7.
762.5207 (calcd. for C,,6H~gOgSlg, 762.5259) .
m/z 762 (2, M}, 747 (4), 705 (2), 633 (5), 632 (18), 25 631 (44), 630 (78), 574 (5}, 541 (10), 540 (18), 494 (9), 438 (2) , 408 (13) , 362 (3) , 308 (2) , 277 (4) , 249 (4) , 207 (4) , 131 (20), 75 (base), 73 (37).
Preparation of analog EV, namely 22-[3-(1'-Methyl-1' hydroxyethyl)phenyl]-23,24,25,26,27-pentanor-la-hydroxyvitamin 30 D3, compound (5c) .
Dienyne 3c (0.020 g, 0.026 mmol) was dissolved in 13 mL
of EtOAc and 42 uL 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 35 hydrogen at room temperature and then filtered through a short column of silica gel. After concentrating the filtrate, the crude residue was purified by flash chromagraphy (1%
EtOAc/hexanes) to afford 17 mg of the mixture of vitamin and previtamin. This mixture was added to 10 mL of isooctane and the solution was heated at reflux for 2 h. After evaporation of solvent, the crude product was dissolved in 0.7 mL of dry THF and 0.17 mL of a THF solution 1 M of tetrabutylammonium fluoride. The mixture was stirred at room temperature for 12 h protected from the light and then 2 mL of a saturated solution of NaCl was added. The mixture was extracted with EtOAc and then the combined organic extracts were dried over MgS04 and concentrated. After passing the residue through a short column of silica gel, the crude product was purified by HPLC (Rainin Dynamax, 1.0 x 25 cm, 8 qua, 100% EtOAc, 4 mL/min) to afford 3.9 mg (32%) of the vitamin 5c as a white, amorphous solid.
Spectral Data: 1~: b 0.58 (3H, C18-Me, s) , 0.82 (3H, CZl-Me, d, J-6.6 Hz), 1.55 and 1.58 (3H and 3H, diastereotopic Me2C, two s), 2.32 (1H, dd, J"6.3 Hz, 13.2 Hz), 2.61 (1H, dd, J"2.7 Hz, 13.2 Hz), 2.83 (1H, br d, J-12.6 Hz), 2.93 (1H, dd, J-2.4 Hz, 13.2 Hz), 4.23 (1H, H3, m), 4.44 (1H, H1, m) , 5.02 (1H, H19, br s) , 5.34 (1H, Hlg, br s) , 6.04 (1H, H~, d, J-11.1 Hz), 6.39 (1H, H6, d, J-11.1 Hz), 7:02 (1H, Ar, d, J-6.9 Hz), 7.26 (3H, Ar, m). The signals at S 2.83 and 2.93 are probably assignable to Hg~ and one of the H22 protons, respectively, or vice versa.
gy: (95% EtOH) ?~"aX 266 nm (E 19, 500) .
~: m/z 464.3307 (calcd. for C31H94O3, 464.3290) .
m/z 464 (14, M), 446 (33), 428 (55), 410 (base), 384 (10), 369 (5), 341 (5), 313 (7), 297 (11), 277 (10), 251 (20), 225 (12), 209 (24), 195 (16), 171 (18), 155 (19), 152 (7), 134 (18), 131 (27) , 105 (25) , 95 (12) , 81 (9) , 69 (5) , 59 (5) .
Scheme B relates to analogs GE and GF described in Examples 4 and 5.

Scheme B
~~OH
v PhZP=O ° ~OH
1 ) n-BuLi or PhLi, ~7B C, THF ~ OH ~ ~H
4)isooctane.
2) ~ H re u~~ w i _.H ~ ~OTIvIS I 3 ~ '~' ~ H
HO
raoMSO'~~ orB~nns 2 4 i H HO'~ OH
O
3) TBAF, THF
1~

Chemi ca1_ Sy)zthesis of Ana1_oa_ GE
This example illustrates preparation of the analog GE, namely 14-Epi-1a,25-dihydroxyvitamin D3 according to Scheme B.
Preparation of 14-Epi-1a,25-dihydroxyvitamin D3 (Analog GE, Compound 3).
To a stirred solution of 1 (67 mg, 0.11 mmol) in anhydrous THF (1.4 mL) at -78°C under argon was added n-butyllithium (74 20~CL, 0.12 mmol, 1.55 M solution in hexanes) to give a deep orange solution. After adding CD ketone 2 (27.1 mg, 0.076 mmol) in dry THF (0.46 mL) , the solution was stirred .for 3 h at -78°C and then warmed to rt. After concentration, the residue dissolved in ether (3 mL) and washed with a saturated solution of NaHC03 (3 mL) and brine (3 mL). After drying (MgS04) and concentrating the ether solution, the crude residue was purified by flash chromatography to afford 48.2 mg (86% yield) of protected vitamin, which was treated with TBAF (0.79 mL, 0.79 mmol, 1 M
solution in THF). After 3 h, the solvent was removed and the 30_crude residue dissolved in EtOAc (5 mL). The solution was washed (water, 3 mL; and brine, 3 mL), dried (NaZS04), filtered and concentrated. Purification by HPLC (50% EtOAc/hexanes, Rainin Dynamax 60 ~r column) afforded after vacuum drying 11 mg (81%) of vitamin 3.
1~ (300 MHZ) : (CDC13) b 0.87 (3H, Czl-CH3, d, J"6.4 Hz) , 0.90 (3H, Cle-CH3, s) , 1.22 (6H, C2s,2-,-CH3, s) , 2.31 (1H, dd, J-13. 3 Hz, 3.5 Hz) , 4.23 (1H, H3, m) , 4.44 (1H, Hl, t, J-5.4 Hz) , 5.00 (1H, H19, br s) , 5.34 (1H, H19, br s) , 6. 14 and 6.33 (2H, H6,~-AB pattern, d, J-11.2 Hz).
.
Ctlemical Synthesi s of Analog GF
This example illustrates preparation of the analog GF, namely 14-Epi-1a,25-dihydroxyprevitamin D3. Preparation of analog GF is seen in Scheme B.
Preparation of 14-Epi-1a,25-dihydroxyprevitamin D3, compound (4).
A solution of vitamin 3 (4.9 mg, 0.012 mmol) in benzene-ds (2 mL) was subjected to three freeze-thaw cycles under vacuum and then placed in a thermostated bath at 80.0°C.
After 4 h, the solution was cooled to rt and the vitamin/previtamin ratio determined by 1H-Nl~t integration (-7:93). The solution was concentrated and purified by HPLC
(100% EtOAc, Rainin Dynamax 60 $~ column) to afford, in order of elution, epi-vitamin 3 (0.3 mg) and epi-previtamin 4 (3.7 mg ) .
1~ (300 MHZ) : (CDC13) b 0.91 (3H, C18-CH3, s) , 0.94 ( 3H, C21-CH3, d, J" 6 . 3 Hz ) , 1. 22 ( 6H, C26,27-CH3 i sj. 1. 75 ( 3H, C19-CH3, br s) , 2.55 (iH, br d, J-16. 6 Hz) , 4.05 (iH, H3, m) , 4.18 (1H, Hi, br s) , 5.65 (1H, H9, m) , 5.80 and 5.85 (H6,~, AB
pattern, d, J"12.5 Hz).
Scheme C relates to analogs HH, HJ and HL described in Examples 6-8.

WO 99116451 g4 PCT/US98I19862 Scheme C
1 ~ OTMS Or OH ~ ~ ( OH
~H
I

OTf ~~~ H 3 2) TBAF, THF
T9DMS0~'OTBDMS pdfPPh ~)2(OAc) .
Cul, ct~NH, DM~
TBDMSO OTBDMS
3) H:, Pd. Lindlar 1 O OH qtnncune, hexenas v I
~OH
O t ( 5) Dess~Matun rOH
penodinane. I H 4) acetone, OH
H heat ~
HO~ ~~ CH~CN
7 ~ (Analog NH) HO' HO~'OH $
61 NaBH,, hleCH , r v ~OH ~OH
OH I I , 7) acetone, H heat ~ H
( H
HO' 9 (Analog HJ) ~~ (Analog HL) 2 O "''' OH Hp' ~ ~'OH
OH t 0) acetone I heat 8) Dess~Martin periodin ane, v~OH v ~OH
CHSCN O \ ~ OHI
(1a,25-(OH)2D9) H 9) Nai3H,, ~ H
HO ~ MeOH HO
Ho'~ off 11 12 chemical S~rntl,pci s of Analoa HH
This example illustrates preparation of the analog HFI, namely 1x,25-dihydroxy-3-epivitamin D3. Preparation of analog HH is according to as seen in Scheme C.
Preparation of 1[3-[(tert-butyldimethylsilyl)oxy)-6,7-dehydro-25-hydroxy-3-epiprevitamin D3 tert-Butyldimethylsilyl ether, compound (3).
To a mixture of enol triflate 2 (80 mg, 0.2 mmol) and lp,3a-enyne 1 (84 mg, 0.22 mmol) in diethylamine (1 mL) and dimethylformamide (1 mL) was added CuI (4.8 mg, 0.003 mmol) sussn~rs sH~r ~RU~ zs) and bis[triphenylphosphine)palladium(II) acetate (5.0 mg, 0.007 mmol}. The reaction mixture was stirred at room temperature for 1.5 h under argon. Ether was added and the mixture was washed with H20 (3 x 5 mL), dried (MgS09) and 5 evaporated in vacuo. The crude dark brown oil was purified by flash chromatography (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.
(300 MHZ) : (CDC13) b 0. 06 (6H, Si-CH3, s) , 0.09 10 (6H, Si-CH3, s) , 0.70 (3H, C18-CH3, s) , 0.88 (18H, Si-t-Bu, two s) , 0.95 (3H, C21-CH3, d, J-6.6 Hz) , 1.21 (6H, C26,2,-2CH3, s) , 1.89 (3H, Cl9-CH3, s) , 2.45 (1H, C14-H, dd, J-16. 5 Hz, 4.5 Hz) , 4 . 0-4 .1 ( 1H, H3, br m) , 4 .18 ( 1H, Hl, m) , 5 . 96 ( 1H, H9, d, J-3.0 Hz).
15 13CNMRNMR (75.5 MHZ): (CDC13} 5 -4.8, -4.7, -4.6, -4.3, 11.1, 18.0, 18.1, 18.7, 19.1, 20.8, 24.2, 25.2, 25.8, 25.9, 28.0, 29.2, 29.4, 35.9, 36.2, 36.4, 39.8, 41.3, 41.9, 44.4, 50.2, 54.7, 64.2, 70.0, 71.1, 88.1, 92.4, 115.5, 122.6, 133.2, 140.3. A satisfactory mass spectrum of this substance could 20 not be obtained. It was best characterized as the corresponding deprotected alcohol.
Preparation of 1(3,25-dihydroxy-6,7-dehydro-3-epiprevitamin D3 compound (4).
To a solution of dienyne 3 (76 mg, 0.12 mmol) in 5 mL THF
25 under argon was added tetrabutylammonium fluoride (0.6 mL of 1.0 M solution in THF, 0.6 mmol). The reaction mixture was stirred at room temperature in the dark for 12 h. It was diluted with ethyl acetate and washed with brine (2 x 10 mL).
The aqueous layer was extracted with ethyl acetate (2 x 10 mL) 30 and the combined organic layer was dried (MgS04) and evaporated in vacuo. Flash chromatography of the residual oil (elution with 5D% ethyl acetate-hexane followed by 90% ethyl acetate-hexane) afforded after vacuum drying 38 mg (76%) of the triol 4 as a colorless oil, which was sufficiently pure 35 for characterization and further reaction.
1H~NM$ (300 MHZ) : (CDC13) b 0.69 (3H, C18-CH3, s) , 0.95 (3H, C21-CH3, d, J-6.6 Hz) , 1.21 (6H, C2s,27-CHs. s) , 1.98 (3H, C19-CH3, br s ) , 2 . 54 ( 1H, Hl4 , dd, J-16 . 0 Hz , 4 . 0 Hz ) , 4 . 04-WO 99116452 PC"T/US98I19862 4 .12 ( iH, H3, br m) , 4 . 23-4 . 28 ( 1H, Hl, narrow m) , 5. 97-5. 98 (1H, H9, narrow m).
isCNMRNMR (75.5 MHZ) : (CDC13) a 11.1, 18.?, 18.8, 20.8, 24.2, 25.2, 28.0, 29.2, 29.3, 35.9, 36.2, 36.4, 39.2, 40.0, 41.9, 44.3, 50.1, 54.7, 63.4, 69.3, 71.1, 87.5, 93.4, 115.2, 122.4, 133.8, 139.4.
uy: (95% EtOH) 1~x 272 nm (e 14,400), 286 nm (E 11,000).
(FAB, NBA matrix) m/z 414.3146 (calcd. for C27H4z03, 414.3134).
M~: (FAB, NBA matrix) m/z 414 (15, M), 413 (11), 397 (base, M - OH), 379 (11), 363 (3), 341 (3), 323 {2), 267 (6), 255 (3), 237 (3), 197 (7), 179 (10), 165 (19).
Preparation of analog HH, 1p,25-Dihydroxy-3-epivitamin D3 , compound ( 6 ) A~stirred mixture of dienyne 4 {27 mg, 0.065 mmol), Lindlar catalyst (27 mg) and quinoline (308 ~ul, 0.17 M in hexanes) in methanol (2.5 mL) was exposed to a positive pressure of hydrogen gas for 22 min. The mixture was filtered and concentrated to afford a residual oil which was purified by flash chromatography (elution with 50% ethyl acetate-hexane followed by 90% ethyl acetate-hexane) to afford 27 mg of the crude previtamin 5. iH-NMR analysis of the latter material showed the complete absence of starting material. A solution of the crude previtamin (27 mg, 0.065 mmol) in acetone (1 mL) was placed in a screw capped vial and heated for 4 h in a constant temperature bath set at 80° C. The residue was concentrated under vacuum and purified by HPLC (85% ethyl acetate-hexane, 4 mL/min, Rainin Dynamax 60A column) to afford after vacuum drying 15 mg (56%) of the vitamin 6 as a colorless oil.
1~$ (300 MHZ) : (CDC13) S 0.54 (3H, Cle-CH3, s) , 0.93 (3H, Czl-CH3, d, J-6.0 Hz) , 1.21 (6H, Czs,z~-CHs. s) , 2.30 {1H, H,a, dd, J-13.0 Hz, 7.5 Hz) , 2.62 (1H, H4a, dd, J-13.0 Hz, 3.7 Hz), 2.82 (1H, H9a, dd, J"11.8 Hz, 3.0 Hz), 4.15-4.30 (1H, H3, 35 m) , 4.40-4.50 (iH, Hi, m) , 5. 00 (1H, H19, narrow m) , 5.32 (iH, H19, narrow ~m) , 6 . O1 and 6 . 39 ( 2H, H6,~ , AB pattern, J" 11. 4 Hz ) .
(75.5 MHZ): (CDC13) b 12.0, 18.8, 20.8, 22.3, 23.6, 27.6, 29.1, 29.2, 29.4, 29.7, 36.1, 36.4, 40.5, 42.8, 44.4, 45.5, 45.9, 56.3, 56.5, 66.8, 71.4, 112.6, 117.0, 125.0, 132.7, 143.3, 147.3.
~$: (CC1,) v 3357 (OH, br s) , 2944 (sp3CH, br s) , 1377 {s} , 1216 (s) , 1053 (s) , 667 (s) cm 1.
jjy: (95% E'tOH) 1~",ax 264 nm (E 17,000) .
m/z 416.3288 (calcd. for CZ~H44O3, 416.3292) .
m/z 416 (21, M) , 398 (72, M - HZO) , 380 (36, M
2H20), 362 (3), 329 (3), 285 (11), 251 (10), 227 (9), 197 (8), 152 (29, A-ring portion after C,,g-cleavage) , 134 (base, m/z 152 - H20) .
E~~AMPLE 7 Chemral SSynthesis of analog HJ
This example illustrates preparation of the analog HJ, namely 1a,25-dihydroxy-3-epivitamin D3. The analog HJ is prepared according to Scheme C.
Preparation of 1-axo-25-hydroxy-3-epiprevitamin D3 compound (7} .
1p,25-dihydroxy-3-epivitamin D3 compound (6), (28.0 mg, 0.067 mmol) was added to the Dess-Martin periodinane reagent (40 mg, o.lo mmol) in dry CH3CN (12 mL). The reaction mixture was stirred at room temperature for 60 min under argon. The resulting bright yellow solution was diluted with ether and washed with a 1:1 mixture (v/v) of saturated aqueous Na2S203 and NaHCO3 solution (20 mL}. The organic layer was then dried (MgS04) and evaporated to dryness. The residue was purified by flash column chromatography on silica gel using 1:3 hexane:ethyl acetate to afford after vacuum drying 25 mg (90%) of 1-oxo-25-hydroxy-3-epiprevitamin D3 as a pale yellow oil, which was sufficiently pure for spectral characterization and further reaction.
{300 MHZ) : (CDC13) b 0.71 (3H, Cle-CH3) , 0.96 (3H, C21-CH3, d, J'6.6 Hz} , 1.21 (6H, CZS,z7-2CH3, s) , 1.78 (3H, Cl9 CH3, s), 2.4-2.6 (1H, m), 2.70-2.85 (1H, m), 4.16 (1H, H3, m), 5.47 (iH, H9, m), 6.05 and 6.11 (2H, H6,~, AB pattern, J'i1.7 Hz) .
j~y: (95% EtOH) lt",a,~ 242 nm (EE 10, 000) , 298 nm (EE 11, 200) .
(CI, NH3) m/z 414.3145 (calcd. for CZ~H42O3~
414.3136).

WO 99/16452 PG"fIUS98119862 (CI, NH3) m/z 415 (15, MH), 414 (7, M), 396 {86, M -H20) , 379 (base, MH - 2 H20) , 363 (4) , 338 (2) , 323 (3) ;- 295 (2) , 267 (10) , 253 (4) , 239 (3) , 213 {6) , 199 (4) , 171 (9) , 157 (6), 135 (3), 121 (4), 107 (3), 95 (6), 81 (4), 69 (2).
5 Preparation of analog HJ, lcx,25-dihydroxy-3-epivitamin D, compound (9) Sodium borohydride (38 mg, 1.0 mmol) was added to an ice cold solution of 1-oxo-25-hydroxy-3-epiprevitamin D3 compound ( 7 ) ( 25 mg, 0 . 06 mmol ) in MeOH ( 2 mL) . After the reaction 10 mixture was stirred for 1 h, tlc (75% ethyl acetate/hexane) showed complete disappearance of starting material. The mixture was extracted three times with ether and the ether extract was dried (MgS04) and then concentrated in vacuo. The crude product was purified by HPLC (10% iPrOH/hexane) to yield 15 17 mg (69%) of the previtamin 8. The latter dissolved in acetone (1 mL) was placed in a screw capped vial and heated for 4 h in a constant temperature bath set at 80°C. The reaction solution was concentrated in vacuo and then the residue was purified by HPLC {10% iPrOH/hexane) to afford 20 after vacuum drying 15 mg (90%) of the vitamin 9 as a colorless oil.
1~I~NM$ (300 MHZ) : (CDC13) S 0.54 (3H, Cle-CH3, s) , 0.93 (3H, C21-CH3, d, J"6.2 Hz) , 1.21 (6H, C26,27-CH3i s) , 2.43 {iH, H4a, dd, J"13.5 HZ, 5.5 Hz), 2.56 (1H, H4a, dd, J"13.5 HZ, 2.9 25 Hz) , 2.83 {1H, H9a, dd, J"11.8 Hz, 3.0 Hz) , 4.0-4.1 (1H, H3, m), 4.25-4.35 (iH, Hl, m), 5.0 (iH, Hi9, narrow m), 5.29 (iH, H19, narrow m), 6.02 and 6.43 (2H, H6,~, AB pattern, J"11.3 Hz).
i3CNMRNMR (75.5 MHZ) : (CDC13) b 12.0, 18.8, 20.8, 22.2, 23.5, 27.7, 29.1, 29.2, 29.4, 36.1, 36.4, 40.5, 40.7, 44.4, 30 45.5, 45.9, 56.3, 56.5, 68.2, 71.1, 73.2, 112.9, 117.0, 125.6, 131.6, 143.2, 147.2.
~$: (CC14) v 3018 (OH, br, s) , 2965 (spa CH, br, s) , 1377 (s) , 1215 (s) , 668 (m) cm 1. l,Zy: (95% EtOH) 1~x 264 nm (e 16,900).
35 ~: m/z 416.3279 (calcd. for CZ~H9lO3, 416.3292) .
m/z 416 (19, M) , 398 (28, M - HZO) , 380 (10, M -2H20), 330 (3), 285 (12), 251 (7), 227 (6), 152 (base, A-ring portion due to C~,B-cleavage) , 134 (73, m/z 152 - H20) , 107 WO 99/16452 PCT/US98/19$62 (26) , 95 (26) , 81 (27) , 55 (30) .
C. hemi~ra~,ynthes~s Df Ana~og~
This example illustrates preparation of the analog HL, namely 1p,25-dihydroxyvitamin D3. Analog HL was prepared according to Scheme C.
1-oxo-25-hydroxyprevitamin D, compound (11) A solution (obtained by gently warming at 35°C the originally obtained suspension) of 20 mg (0.05 mmol) of 1a,25 dihydroxyvitamin D3 (10) in 4 mL of anhydrous CH3CN was added dropwise to a well stirred suspension of,Dess-Martin reagent (26 mg, 0.065 mmol) in CH3CN (4 mL) under argon at room temperature. After 60 min stirring at room temperature, an additional 6 mg (0.3 molar equivalents) of oxidant was added in one portion and stirring was maintained for another 60 min.
Ether (10 mL) was added and the resulting mixture was washed with a 1:1 mixture of saturated aqueous NaZS203 and NaHC03 solution (20 mL). The organic layer was then dried (MgS04) and evaporated to dryness. The residue was purified by flash column chromatography on silica gel using hexane: ethyl acetate (1:3) to afford 17.5 mg (88% yield) of 1-oxo-25-hydroxyprevitamin D3 (11). This substance was prepared in lower yield (<40%) using Mn02.
1H~: (300 MHZ) : (CDC13) b 0.72 (3H, C18-CH3, s) , 0.97 (3H, CZl-CH3, d, J-6.6 Hz) , 1.23 (6H, Cz6,2~-2CH3, s) , 1.80 ( 3H, Cl9-CH3, s ) , 4 .17 ( iH, H3, m) , 5 . 50 ( iH, H9, m) , 6 . 04 and 6.14 (2H, H6,~, AB pattern, J'11.7 Hz).
13C~NMR: (75 MHZ) : (CDC13) a 11.2, 11.7, 18.7, 20.8, 23.3, 25.I, 28.4, 29.2, 29.3, 35.9, 36.1, 36.4, 38.8, 42.1, 44.4, 47.0, 50.6, 54.3, 67.0, 71.1, 71.2, 127.3, 132.5, 134.1, 136.4, 151.2, 197.7.
yy: (95% EtOH) 1~X 240 nm (E 15,000), 300 nm (E 11,800);
(ether) 1~X 234 nm (E 15,100), 288 nm (E 11,200).
Preparation of analog HL, 1,25-Dihydroxyvitamin D3, compound (13) Sodium borahydride (38 mg, 1.0 mmol) was reacted with 1-oxo-25-hydroxyprevitamin D, (li) (25 mg, 0.06 mmol) in MeOH (2 mL) and then worked up as described for the preparation of the la,3a-diastereomer 8. The product was purified by HPLC
(10% iPrOH/hexane) to yield after vacuum drying 17 mg (69%) of the previtamin 12. The latter was dissolved in acetone (1 mL) and placed in a screw capped vial and heated in a constant temperature bath set at 80°C for 4 h. It was concentrated in vacuo and purified by HPLC (80% EtOAc/hexane) to afford after vacuum drying 12 mg (70%) of the vitamin 13 as a colorless oil.
'~ (300 MHZ) : (CDC1~) b 0.55 (3H, C19-CH3, s) , 0.94 (3H, C2~-CHI, d, J'5.7 Hz) , 1.22 (6H, CZS,Z-,-CH3, s) , 2.50 (2H, m), 2.83 (1H, m), 4.11 (1H, m), 4.36 (1H, m), 5.01 (iH, H19.
d, J'1.5 Hz), 5.29 (1H, H19, d, J'1.2 Hz), 6.05 and 6.45 (2H, H;," AB pattern, J'i1.4 Hz).
(100$ EtOH) 1,",aX 264 nm (e 17, 100) .
Scheme D relates to analogs HQ and HR described in Examples 9 and 10.
2 O H I ICOTaOMSi ,~OT80M5 tf !. H ..
\ 1 I~_~ 2a,b H
-~ 3a.b 2~ H~, ?d. l.inGlar .Isc aTaoMS V 1 ?~IPPn~h(Cat12. cumcana. naxana$ I OT3GM5 - ' Cul. ~S=NH~ObI~
2 5 ~30MSO~~OTBDMS H
=-C(CH~)=oTBDMS: ~ . H TaoMsa ~\ -. -C(CH~ZOToDMS: R = H » I~OH
TaDMSO
H 3llsooeune. 4a,b retlux ~; TaAF. ~~ I~tan~~ H H
j.l THF ndux \
5a (Analog HQ) sf TaAF. '~ ~
THF ~H
HC-OOH
H
Sb (Analog HR) NC~
~hem,i-,,r,a1_,~ ant e~i s of Analog HO
Thus example illustrates preparation of the analog HQ, namely (22S)-1a,25-Dihydroxy-22,23,23,24-tetradehydrovitamin suBSTrrurs sHEEr ~RUt.~ zs~

D3. Analog HO is prepared according to Scheme D.
Preparation of (22S)-1a,25-di(te-rt-butyldimethylsilyloxy)-6,7,22,23,23,24- hexadehydro-previtamin D3 tert-butyldimethylsilyl ether, compound (3a).
Bis(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 (MgS04), 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 ,um, 1% ethyl acetate/hexanes) to afford after drying, spectroscopically homogeneous dienyne 3a (59 mg, 75%) as a colorless oil.
- 1~: b_0.06 (6H, Si-Me2, s), 0.07 (6H, Si-Mez, s), 0. 09 (6H, Si-Me2) , 0.72 (3H, Cle-Me, s) , 0.85 (9I~, t-Bu, s) , 0.88 (9H, t-Bu, s) , 0.89 (9H, t-Bu, s) , 1.09 (3H, C21-Me, d, J-6. 6 Hz) , 1.30 (3H, C26,27-~3. s) , 1.31 (3H, 026,2.,-CH3, s) , 1.90 (3H, Cl9-Me, br s), 4.09 (iH, H3, broad m, W-15 Hz), 4.19 (1H, Hl, m), 5.18 (iH, H22, dd, J-6.6 Hz, 6.6 Hz), 5.28 (iH, H2q, dd, J-6.6 Hz, 1.8 Hz), 5.97 (1H, H9, narrow m). .
Preparation of (22S)-1x,25-Di(tert-butyldimethylsilyloxy)-22,23,23,24-tetradehydro-previtamin D3 tert-butyldimethylsilyl ether, compound (4a) A mixture of dienyne 3a (10.0 mg, 0.013 mmol), quinoline (75 ~L, 0.17 M in hexanes, 0.013 mmol) and Lindlar catalyst (21 mg) in hexanes (3.5 mL) was stirred under an atmosphere of hydrogen for 1 h. The mixture was filtered through a short pad of silica gel and the residue concentrated to afford a colorless oil. The crude product was purified by HPLC (Rainin Dynamax, l.0 x 25 cm, 8 ~Cm, 0.1% ethyl acetate/hexanes) to afford after vacuum drying, the spectroscopically pure previtamin 4a (8.0 mg, 81%) as a colorless oil.
S 0.05 (3H, Si-Me, s), 0.06 (3H, Si-Me, s), 0.07 (6H, Si-Me2, s) , 0.09 {6H, Si-Me2, s) , 0.71 (3H, C,8-Me, s) , 0.85 (9H, t-Bu, s), 0.886 (9H, t-Bu, s), 0.895 (9H, t-Bu;-s), 1. 09 (3H, C21-Me, d, J-6.6 Hz) , 1.30 (3H, Cz6,2~-Me, s) , 1.31 (3H, C2s,27-Me, s) , 1.65 {1H, Cl9-Me, br s) , 4. O1-4.10 (1H, H3, m) , 4.11 (1H, Hl, br s) , 5.17 (1H, H22, dd, J-6. 9 Hz, 6.9 Hz) , 5.27 (1H, H24, dd, J-6.6 Hz, 1.8 Hz), 5.55 (iH, H9, narrow m), 5.73 and 5.88 (2H, H~ and H~, AB pattern, J"12.0 Hz).
Preparation of analog HQ, (22S)-1a,25-dihydroxy-22,23,23,24-tetradehydrovitamin Dj, compound (5a) A solution of previtamin 4a (12.0 mg, 15.9 mmol) in isooctane (8.0 mL) was refluxed (-100°C) under an argon atmosphere for 2.4 h. The solvent was removed under vacuum to afford a colorless residue, which was determined to be a 88:12 inseparable mixture of vitamin and previtamin. A
solution of this mixture in THF (1.0 mL) was treated with tetra-butylammonium fluoride (275 ~L, 1.0 M in THF, 0.275 mmol) at room temperature for 15 h, protected from the light.
The reaction was quenched by the addition of brine (2 mL) and the mixture was extracted with ethyl acetate (4 x 2 mL). The combined organic extracts were dried (MgS04) and concentrated and the crude product passed through a short pad of silica gel. Purification was effected by HPLC (Rainin Dynamax, lx cm, 8 E,cm, 4 mL/min, 100% ethyl acetate) to afford after drying 4.7 mg (71%) of the vitamin (5a) as a viscous colorless 25 oil. 1~: s-0.57 (3H, C18-Me, s) , 1.08 (3H, Czl-Me, d, J-6. 6 Hz) , 1.34 (6H, Cz6,27-2~3r s) r 2.32 (1H, H~a, dd, J-13.2 HZ, 6.0 HZ), 2.60 (1H,.H9a, dd, J"13.2 HZ, 3.0 HZ), 2.83 (1H, H9p, dd, J'11.7 Hz, 3.0 Hz) , 4.23 (1H, H3, m, W-20 HZ) , 4.43 (1H, HI, m, W-12 Hz) , 5.00 (1H, Hl9Z, narrow m) , 5.33 (1H, Hlser narrow m), 5.28-5.35 (2H, HZZ and H29, m, partially obscured by HlgE) , 6.02 and 6.38 (2H, H6 and H~, AB pattern, J-11.2 Hz) .

Chemical SynthPSis of Analog HR
This example illustrates preparation of the analog HR, namely (22R)-1a,25-dihydroxy-22,23,23,24-tetradehydrovitamin D3. Analog HR was prepared according to Scheme D.
Preparation of (22R)-1a,25-di(tert-butyldimethylsilyloxy)-6,7,22,23,23,24- hexadehydro-previtamin D3 tert-butyldimethylsilyl ether, compound (3b) Bis(triphenylphosphine)palladium(II) acetate (6.0 mg,w 8.1 mmol) and copper(I) iodide (5.8 mg, 30.4 mmol) were added at ambient temperature to a mixture of enol triflate 2b (64 mg, 0.123 mmol), enyne 1 (56 mg, 0.147 mmol) in DMF (1.2 mL) and diethylamine (1.2 mL) under an argon atmosphere. The mixture was stirred at room temperature for 2.5 h after which ether (10 mL) was added and the mixture washed with brine (3 x 10 mL). The organic layers was dried (MgS04), filtered and concentrated to afford a dark brown residue. Purification was effected by a short path flash chromatography (silica gel, 15%
ethyl acetate/hexanes) followed by HPLC separation (Rainin Dynamax, 1.0 x 25 cm, 8 ~,cm, 1% ethyl acetate/hexanes) to afford after drying, spectroscopically homogeneous dienyne 3b (86 mg, 93%) as a colorless oil.
b_0.06 (6H, Si-Me2, s) , 0. 07 (6H, Si-Me2, s) , 0.09 (6H, Si-Me2, s), 0.72 (3H, Cle-Me, s), 0.85 (9H, t-Bu, s), 0.88 (9H, t-Bu, S) , 0.89 (9H, t-Bu, S) , 1. 09 (3H, C21-Me, d, J'6.6 Hz) , 1.29 (3H, Cy6,27-~3. s) . 1.30 (3H, C26,2~-CH3, s) , 1.89 (3H, C19-Me, br S) , 4.1 (1H, H3, br m) , 4.19 (1H, iii, m) , 5.15 (iH, H22, dd, J"6.6 Hz, 6.6 Hz) , 5.27 (1H, H24, dd, J"6.6 Hz, 1.8 Hz), 5.97 (iH, H9, narrow m).
Preparation of (22R)-1a,25-di(tert butyldimethylsilyloxy)-22,23,23,24-tetradehydro-previtamin D3 tert-butyldimethylsilyl ether, compound (4b) A mixture of dienyne 3b (10.0 mg, 0.013 mmol), quinoline (80 E,cL, 0.17 M in hexanes, 0.013 mmol) and Lindlar catalyst (20 mg) in hexanes (3.0 mL) was stirred under an atmosphere of hydrogen for 40 min. The mixture was filtered through a 3o short pad of silica gel and the residue concentrated to afford after drying, a colorless oil. HPLC separation (Rainin Dynamax, 1.0 x 25 cm, 8 E.cm, 0.1% ethyl acetate/hexanes) afforded the spectroscopically pure previtamin 4b (7.0 mg, 70%) as a colorless oil.
1~: b 0.05 (3H, Si-Me, s), 0.06 (3H, Si-Me, s), 0.07 (6H, Si-Me2, s) , 0.09 (6H, Si-Me2, S) , 0.71 (3H, C18-Me, s) , 0.85 (9H, t-Bu, S), 0.886 (9H, t-Bu, S), 0.894 (9H, t-Bu, S), 1. 09 (3H, C21-Me, d, J-6.6 Hz) , 1.29 (3H, C2s,2~-CH3, s) , 1.31 (3H, CZ6,2~-CHs. s) , 1.65 (3H, C19-Me, broad s) , 4.01-4.10 (1H, H3, m) , 4.11 (1H, H1, broad s) , 5.14 (iH, H22, dd, J'6.6-- Hz, 6 . 6 Hz ) , 5 . 2 7 ( 1H, H2, , dd, J' 6 . 6 H2 , 2 . 1 Hz ) , 5 . 54 ( iH, H9, narrow m) , 5.72 and 5.90 (2H, H6 and H~, AB pattern, J'12.0 Hz) .
Preparation of analog HR, (22R)-1a,25-dihydroxy-22,23,23,24-tetradehydrovitamin D3, compound (5b).
A solution of previtamin 4b (15 mg, 19.9 mmol) in isooctane (10 mL) was refluxed (-100°C) for 2 h under an argon atmosphere. 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. The reaction was quenched by the addition of brine (2 mL) and then the mixture was extracted with ethyl acetate (4 x 2.0 mL). The combined organic extracts were dried (MgS04), filtered and concentrated.
Purification was effected by short column flash chromatography (silica gel, 100% ethyl acetate) followed by HPLC separation (Rainin Dynamax, 100% ethylacetate) to afford after vacuum drying vitamin 5b (5.4 mg, 66%) as a colorless foam.
b 0 . 57 ( 3H, C18-Me, s ) , 1. 09 ( 3H, C21-Me, d, J' 6 . 6 Hz) , 1.34 (6H, CZS,27-2CH3, s) , 2.32 (1H, H4p, dd, J'13 .2 Hz, 6. 0 HZ) , 2.60 (1H, H9a, dd, J'13.2 HZ, 3.0 HZ) , 2.83 (1H, H9p, dd, J'12.0 Hz, 3.0 Hz), 4.23 (1H, H3, m, W'20 HZ), 4.43 (1H, Hl, m, W'12 Hz) , 5.00 (1H, Hl9z, s) , 5.33 (1H, H19E, s) , 5.26-5.35 (2H, H22 and H24, m, partially obscured by Hl9e) . 6.02 and 6.38 3 0 ( 2H, H6 and H~, AB pattern, J' 11. 2 Hz ) .
Scheme E relates to the analog HS described in Example Il.

Scheme E
_ ,~~o ~.
p t) n~au4 Or PItU. ~T9 °C. TNF I ' i OTVtS
1 ~ 2I
a~0 , H 31 LiAIHy. lti~lr raoMSa~ oTaoMS .-H orMS ~ , s I
p H T30MS0'~~pTBDMS TdOMSO'~
1 O Hp a) T3aF. THF
OH
N
5, ~ Analog ht5 HO' EKAMpLE 1'1 chemical synthesis of Analoq HS
This example illustrates preparation of the analog HS, 20namely 1a,18,25(OH)2D3, as seen in Scheme E.
Preparation of 18-acetoxy-25-trimethylsilyloxy-la-tert-butyldimethylsilyloxy- vitamin D3 tert-butyldimethylsilyl ether, compound (3).
A solution of A-ring phosphine oxide 1 (122 mg, 0.21 mmol) in dry THF (3 mL) was treated with n-butyllithium (0.14 mL, 0.21 nnaol, 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.
1~: (cDCl,) s o.07 (12H, si-Me, series of s), o.lo (9H, TMS), 0.87 (9H, t-Bu, s), 0.89 (9H, t-Bu, s), 1.03 (3H, fyl-CHg, d, J-4.0 Hz) , 1.20 (6H, C26,27-CH3. s) , 2.01 (3H, Ac, s) , 2.87 (1H, H9a, d, J'12.8 Hz) , 3.86 (2H, 2H18, s) , 4.1-4.3 (iH, H3, m) , 4.37 (iH, Hl, apparent t, J~4.9 Hz), 4.86 (iH, Hf9, d, J-1.9 Hz), 5.18 (1H, H19, br s) , 6.03 and 6.19 (2H, Hs,~, AB pattern, d, J-11.1 Hz).
Preparation of 18-hydroxy-25-trimethylsilyloxy-la-tert-butyldimethylsilyloxy-vitamin D3 tert-butyldimethylsilyl Ether (4) .
SUBSTITUTE SHEET (RULE 26) ethyl ether (0.2 mL) and was added dropwise to a solution of LiAlH4 (21 mg, 5.4 mmol) in ether (0.5 mL). The reaction mixture was stirred for 30 minutes, by which time the solution had become viscous and an additional 0.2 mL of ether was added. After stirring for 20 minutes, the reaction mixture was quenched with ethyl acetate (1 mL) and then filtered through a sintered glass funnel. The grey solid was washed with ethyl acetate (5 mL) and the combined filtrate concentrated. The crude residue was purified by flash chromatography (20% ethyl acetate/hexanes) to afford, after vacuum drying, 102 mg (78%) of the protected alcohol precursor, compound 4.
The analytical data for the precursor is:
(30o MHz): (cncl3) s o.06 (12H, si-cH" s), o.lo (9H, TMS, s), 0.87 (9H, t-Bu, s), 0.89 (9H, t-Bu, s), 1.04 (3H, C2i, CH3, d, J ' 6. 3 Hz) , 1.20 (6H C26,2~-CHs. s) , 0. 9-2.5 (remaining ring and side chain hydrogens, series of m), 2.88 (1H, br d, J ' 11.8 Hz), 3.44 (1H, H18, d, J ' 11.5 Hz), 3.53 (1H, Hlg, d, J ' 11.5 Hz) , 4.18 (1H, H3, m) , 4.37 (iH, Hl, m) , 4.84 f (1H H19, br s) , 5. 18 (iH, H19, br s) , 6.04 and 6.22 (2H, H6, ~ AB pattern, d, J ' 11.1 Hz ) .
13CNMR (75.5 MHZ): (CDC3) S -5.1, -4.8, -4.7, 2.6, 18.1, 18.2, 19.3, 20.7, 22.0, 23.9, 25.8, 25.9, 27.6, 28.8, 29.8, 30.0, 35.7, 36.1, 36.6, 44.8, 45.3, 46.0, 49.7, 55.3, 56.9, 61.5, 67.5, 72.0, 74.1, 111.3, 118.1, 122.8, 135.9, 141.0, 148.3.
~$: (CC14) v 3500 (OH, br) , 2960 (C-H, s) , 2930 (C-H
s), 2860 (C-H, m), 1650 (w), 1470 (w), 1360 (w), 1250 (s), 1090 (s), 1045 (s), 910 (m), 840 (s) cm 1.
~: (95% EtOH) A",aX 264 nm (e 18,000) : ?~",in 232 nm (E
10, 900) .
calcd. for Cq2Hg0~4Si3: 68.79; H, 11.00. Found: C, 68.74; H, 11.17.
Preparation of 1a,18,25(OH)ZD3, compound 5.
The analog HS (5) was prepared by adding tetra-n-butyl-ammonium fluoride (2.16 ;CL, 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 WO 99116452 PC'T/US98/19862 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, 2.0 x 25 cm, 8 ~.m silica column, EtOAc) to give, after vacuum drying, the analog HS (5, 7 mg, 70%) as a white foam.
The analytical data for the analog HS (5) is:
(300 MHZ) : (CD30D) b 1.07 (3H, C21-CH3, d, J - 6.4 Hz) , 1.16 (6H, C26,2-,-CH3, s) , 1. 0-2. 2 (remaining ring and side chain hydrogens, series of m), 2.24 (1H, dd, J ~ 13.2 Hz, 7.2 Hz), 2.51 (2H, br d, J - 13.0 Hz), 2.91 (1H, br d, J - 11.2 Hz) , 3.35 (2H, H18, d, J - 11.8 Hz) , 3.41 (iH, H18, d, J - 11.8 Hz), 4.10 (iH, H3, m), 4.34 (1H, Hl, t, J - 5.6 Hz), 4.87 (iH, H19, s) , 5.28 (1H, H19, s) , 6.06 and 6.32 (2H, H6,~, AB pattern, d, J " 11.1 Hz).
lly: (95% EtOH) J~",X 264 nm (E 18, 100) : h",in 230 rim (E
10, 300) .
,: m/z 432.3242 (calcd. for C27H9,,O9, 432.3241) .
m/z 432 (1, M), 414 (4, M - H20), 396 (1, M - 2H20), 257 (2), 171 ( 3 ) , 152 ( 1, A-ring fragment due to C~, a cleavage) , 13 4 ( 8 , 152 - HZO), 105 (6), 91 (10), 79 (17), 69 (20), 59 (base).
Scheme F relates to the analog IB described in Example 12.

WO 99/16452 1 ~8 PCTNS98119862 Scheme F
i ~ w i ' coots. ~ ~ cy ~~
i ~ F, II 1 .~ o ~~GOOMa ~1 P:fPP1>>~(CiIT. ~ 2) ?CC. P7Fa OI HN ~ Z Cui, EtZNhI, MF CN G, I

H ~ 4 _ OH O ri 1~ ) + n~outi. ~T8 °C.
Trig )TBOMS
T30MS0~

Chemical Svn hesis of ~naloa IB
This example illustrates preparation of the analog IB, namely 23-(m-dimethylhydroxymethyl)phenyl)-22-yne-24,25,26,27-tetranor-la(OH)D~, as seen in Scheme F.
Preparation of 23-[3-(1'-methyl-1'-hydroxyethyl)phenyl]-22,23-tetradehydro-24,25,26,27-tetranor-la-OH-D~.
In step 1, 1 and 2 are reacted in the presence of palladium(0) resulting in 3, which was obtained pure by flash chromatography using the solvent 20% ethyl acetate in hexane.
In step 2 , 55 mg of the product of step 1 was reacted with 183 mg pyridinium chlorochromate (PDC), 12 mg pyridinium trifluoroacetate (PTFA) and 100 mL CHZC1~ according to a standard procedure. The reaction was carried out at room temperature for 5 hours. The resulting black mixture was filtered and washed with CH2ClZ and extracted with ethyl suesn~ur~ sHe~ ~RU~ zs) NOT TO BE TAKEN INTO ACCOUNT FOR THE PURPOSE OF INTERNATIONAL
PROCESSING
NO TENER EN CUENTA A LOS EFECTOS DE LA TRAMITACIUN INTERNACIONAL
NE PAS PRENDRE EN COMPTE AUX FINS DU TRAITEMENT INTERNATIONAL

WO 99116452 ~ ~ ~ PCTIUS98/19862 Scheme G
HO OH~i HO o ~ ~ . H ~ .~ 9a,10a i i w ' t) hv, a50 :vaa Hancvia lamp, p~rex. MeOH ~ HO 2, Analog JM
HO OH ' I ~ ~ H ~ H g(i.l0a ; Hod 3~ Analog JN
OH HO OH~
9a,10c~
2) 1,0 °C. OMF. ~ ~ H ~ li oase, to hours i HOB 5~ Analog JO i OI' 4 ~ (ta.25-(OH)ZD~) ~ .~. _ Ho'~~oH ~ Ho i 9(i,10p ~H I~ ~ 6, Anaiog JP
~HO
E~LAMpLE I3 Chem,'_ca~ ~rnthes,'_~ of AnalogWlH
This example illustrates preparation of the analog JM, namely 1a,25-Dihydroxy-7-dehydrocholesterol, 9a,10ø-isomer, as seen in Scheme G.
After 1a,25-dihydroxyprevitamin D, (1) (120 mg) in methanol was saturated with argon for 1 h, the solution was photochemically irradiated (Hanovia 450 watt medium pressure mercury lamp, pyrex filter, 1~ > 300 nm) for 3 h at room temperature. The solution was concentrated and subjected to HPLC (Raining Microsorb, 5 ~m silica, 10 mm x 25 cm, 11%
isopropanol/hexanes) to afford in order of elution JM (2) (9.1 mg, 7.6%), JN (3) (15.0 mg, 12.5%) and the starting previtamin (10.6 mg, 8.8%). Analysis of the crude mixture by 'H-NMR
spectroscopy showed the ratio of JN:JM to be 3:1. Data for analog JM:
z~ (300 MHZ) : (CDC13) b 0.63 (3H, C1g-CHj, s) , 0.95 SUBSTITUTE SHEET (RULE 26) (3H, 019-CH3, s) , 0.96 (3H, C2~-CH3, d, J-5.6 Hz) , 1.22 (6H, 026,2'7-CH3i s) , 0.85-2.2 (remaining ring and side chain hydrogens, various m), 2.35 (iH, apparent t, J-12.7 Hz), 2.55 {1H, d with fine structure, J'14.2 Hz), 2.70 (1H, m), 3.77 (1H, Hl, br s) , 4.07 (1H, H~, m) , 5.38 (1H, Hs or 7, ddd, J"5.5 HZ, 2.8 HZ, 2.8 HZ), 5.73 (1H, H~ orsi dd, J-5.5 HZ, 2.2 HZ).
130-C-NMR (75~5 MHZ): (CDC13) S 11.9, 16.3, 18.8, 20.8, 20.9, 23.0, 28.1, 29.2, 29.4, 36.1, 36.4, 38.0, 38.5, 39.2, 40.0, 43.1, 44.4, 54.7, 55.8, 65.5, 71.1, 73Ø, 115.2, 122.1, 141.4. ~y: (100% EtOH) 1~",aX 294 nm (E 8,400) , 282 nm (e 13 , 400) , 272 nm (e 12, 800) ; l~,pi" 290 nm (E 7, 800) , 278 nm (E
11, 500) ; hg,, 264 nm (e 9, 600) .
(CI, CH4) m/z 417.3365 (calcd. for CZ~H44~3 plus H, 417.3370).
~: (CI, CH4) m/z 417 (28, Ngi), 400 {67), 381 (31), 354 (11), 338 (6), 323 (6), 297 (4), 267 (4), 251 (8), 225 (10), 211 (10) , 197 (11) , 171 (19) , 157 (15) , 119 (12) , 105 (15) , 91 (14), 81 (14), 69 (27), 59 (base).
FXAMPT.E 14 " ; ":~a' S.nthes~s of Ana~oa JN --This example illustrates preparation of the analog JN, namely analog JN, 1a,25-Dihydroxylumisterol, 9~,10a-Isomer (3), as seen in Scheme G.
Analog JN (3) is prepared similarly to and accompanies preparation of the analog JM (2) in the synthesis described in Example 13. The spectroscopic data for JN are as follows.
(300 MHZ) : (CDC13) S 0.61 (3H, Cis-CH3, s) , 0.78 ( 3H, Cl9-CH3, s ) , 0 . 91 ( 3H, 021-CH3, d, J" 5 . 2 Hz ) , 1. 21 ( 6H, C2s,2~-CHs. s) , 0.70-2.30 (remaining ring and side chain hydrogens, various m), 2.50 (2H, m), 4.10 (1H, Hl, dd, J-9.2 Hz, 4.8 Hz), 4.14 (iH, H3, dd, J-3.0 Hz, 3.0 Hz), 5.45 (1H, Hs or m m) ~ 5~75 (1H, H7 or s~ dd, J-5.1 HZ, 1.7 HZ) .
1'C~NMg (75,5 MHZ): (CDC13) b 7.4, 18.3, 18.5, 20.9, 21.7, 22.6, 28.8, 29.2, 29.4, 29.7, 36.2, 37.5, 38.9, 39.5, 41.4, 43.9, 44.4, 46.2, 49.5, 57.3, 66.2, 71.1, 75.8, 115.5, 123.6, 137:2, 142.2.
uy: (100% EtOH) ~ 282 nm (E 6,900), 274 nm (E 7,300);
~,'h 2g4 nm (e 3,900), 264 nm (E 5,900).

~: m/z (CI, CH4) 417.3365 (calcd. for CZ~H,qO3 plus H, 417.3370). ._ (CI, CH4) : m/z 417 (86, MH) , 400 (base) , 382 (60) , 366 (13), 343 (8), 325 (6), 311 (5), 287 (15), 269 (13), 251 5 (9) , 227 (13) , 213 (9) , 174 (46) , 157 (21) , 143 (14) , 119 (7} , 105 (8), 95 (8), 81 (8), 69 (14), 59 (38).

Chemical Synthe~ia of Analog JO
This example illustrates preparation of the analog JO, 10 namely 1a,25-dihydroxypyrocholecalciferol, 9a,10a-isomer (5), as seen in Scheme G.
An argon flushed solution of 1a,25-dihydroxyprevitamin D3 (1) (54.2 mg; or 1a,25-dihydroxyvitamin D3 {2) may be used) dissolved in DMF (15 mL) containing a drop of 2,4,6-15 trimethylpyridine was heated in a screw cap vial (156 °C) for 18 h. The cooled solution was then concentrated and the crude residue was purified by HPLC (Rainin Microsorb, 5 ~Cm silica, mm x 25 cm, 11% isopropanol/hexanes) to afford in order of elution analog JP (6} (7.3 mg, 13.5%), analog JO (5) (20.1 mg, 20 37.1%) and 1a,25-dihydroxyvitamin D3 (2.1 mg, 3.9%~-. Analysis of the crude mixture by 'H-NMR spectroscopy showed the ratio of JO to JP to be 3:1.
Data for analog JO:
'H~NM$ (300 MHZ) : (CDC13} b 0.53 (3H, Cle-CH3, s) , 0.90 25 {3H, C21_CH3, d, J'6.0 Hz) , 1.02 (3H, Ci9-CH3, s) , 1.21 (6H, 026,27-~3. s} . 0.80-2. 05 (remaining ring and side chain hydrogens, various m), 2.15 (iH, dd, J-12.6 HZ, 7.6 Hz), 2.26 (1H, d with fine structure, J-6.1 Hz), 2.54 (1H, br, d, J"6.1 Hz) , 4.16 (iH, H3, dddd, J"2.8 Hz, 2.8 Hz, 2. 8 Hz, 2. a Hz} , 30 4.31 (1H, Hl, dd, J-12. 0 Hz, 4.6 Hz) , 5.34 (1H, Hs or 7, d, J-5.7 HZ), 5.61 (1H, H~ or sr dd, J-5.7 HZ, 2.5 HZ).
"C-NMRNMR (75.5 MHZ): (CDC13) b 12.2, 17.4, 18.7, 20.8, 20.9, 26.0, 28.5, 29.2, 29.4, 29.7, 36.2, 36.4, 37.6, 38.0, 41.1, 44.4, 48.7, 50.6, 56.4, 57.6, 66.7, 66.9, 71.1, 111.7, 35 121.1, 134.8, 140.1.
IIy: (100% EtOH) 1~",a,~ 286 nm (E 9, 400) , 276 nm (e 9, 300) ;
h",i" 280 nm (E 8, 800) ; 1~,,, 296 nm (e 5, 700) , 266 nm (e 7, 000) .
(CI, CH4) m/z 417.3366 (calcd, for C2~HqqO3 plus H, 417.3370) . ~: (CI, CH4) m/z 417 {49, lei) , 400 (base) , 382 (54) , 364 (9) , 343 (4) , 326 (4) , 312 (3) , 287 (4) , 269- -(4) , 251 (4), 227 (6), 213 (4), 197 (6), 157 (12), 143 (8), 111 {9), 95 {13), 81 (17), 69 (24}, 59 (85).
' ' Chemical Synthesis of Analog JP
This example illustrates preparation of analog JP, namely JP, 1a,25-dihydroxyisopyrocholecalciferol, 9,10(3-isomer (6), as seen in Scheme G.
l0 Analog JP (6) accompanies preparation of JO (5) in the synthesis described in Example 15. The spectroscopic data for JP follows.
Data for analog JP:
(300 MHZ) : (CDC13) a 0. 65 (3H, Cle-CH3, s) , 0.92 (3H, C21-CH3, d, J'5.3 Hz) , 1.21 (6H, C26,27-CH3, s) , 1.30 (3H, Clg-CH3, s) , 0.80-2. 08 (remaining ring and side chain hydrogens, various m), 2.39-2.66 (3H, overlapping m), 3.71 (1H, H1, dd, J'2.8 Hz, 2.8 Hz), 3.94 {iH, H3, dddd, J-10.9 Hz, 10.9 Hz, 5.5 Hz, 5.5 Hz) , 5.34 (1H, H6 or -,, ddd, J-5.5 Hz, 2.7 HZ, 2.7 HZ) , 5.95 (1H, H~ ar 6, d, J~5.5 HZ) . -13s-NMR (75.5 MHZ): (CDC13) S 18.3, 18.6, 20.4, 20.9, 22.4, 26.1, 28.8, 29.2, 29.3, 29.7, 36.1, 37.5, 39.2, 41.2, 42.0, 43.5, 44.4, 49.2, 57.3, 69.8, 71.1, 74.5, 115.2, 122.8, 135.5, 142.8.
IIY: (100% EtOH} 1~"ax 286 nm (E 7, 800) , 278 nm (E 7, 900) ;
1~~,, 296 nm (e 5, 200) , 270 nm (e 6, 500) .
(CI, CH4) m/z 417.3351 (calcd. for C2~H4qOg plus H, 417.3370).
(CI, CH4) m/z 417 (36, lei) , 400 (base) , 382 (51) , 364 (12), 342 (4), 312 (3), 288 (6), 270 (10), 252 (10), 215 (9) , 197 (6) , 171 (11) , 157 (7) , 143 (5) , 123 (6) , 105 (13) , 91 (8} , 81 (8) , 69 (17) , 59 {40) .
Scheme H relates to analogs JR, JS, JV and JW described in Examples 17-20.

Ss~hem~H
R. A
- a) Sml,,, THF. ) ' iil 31 Buli: PdfFSPh 6'~~ ty EcLi.O°C P~COC. ~ iPrOH 1)a. I
19 a PhC00 !! H
a va Zi R. T. .F, r; 1" 5) T2AF, Ti-iF ' T aOMSO-~3~OT2DMS 1 '~ C ~ D ~ ~ ( I I~CH
5~~~1 fi2 3 8 i1 H
O ether, 0-25 °C T BOMSO--SOT BOMS pH ~S~) t I R' _ --~OThIS
7) Nacnthalene- 61 hv, a5o war.
Cr(CO)~. ; Har:ovia Iamp, id ~ R'- '-~OH 1 acetone.40°C j quar;z.
I MtOH
t R
R
SOZ ADDUCTS A AND B fi) SC, I H H
,i. ~ -r't 7a, 7b I ~ 6, JR \j S, JW
9) neat. NaHCOg, HO'-~OH OH (6Ct) ethSncl R
I H
8. JS
~
HO-- "OH
This example illustrates preparation of analog TR, namely 7,8-cis-1a,25-dihydroxyvitamin D3 as seen in Scheme H.
Preparation of analog JR, 7,8-cis-1a,25-dihydroxyvitamin D3.
To the vinylallene triol 4 (19.7 mg, 0.047 mmol) and (ry6-naphthalene) tricarbonylchromium (14.7 mg, 0.0557 mmol) in a 10 mL flask with a stir bar was added 1 mL of acetone (distilled from CaS04). After deoxygenation of the mixture by four freeze-pump-thaw cycles, the solution was stirred at 40°C
under a positive pressure of argon for 4 h. Acetone was removed under reduced pressure and the product was purified by flash chromatography (silica gel, 80% ethyl acetate/hexanes) followed by separation by HPLC (80% ethyl acetate/hexanes, Rainin Microsorb column, 4.0 mL/min flow rate) to afford three components in the following order of SUBSTITUTE SHEET (RULE 26) elution: major product A (17.0 mg, 86.4%), recovered starting material B (1.4 mg, 7.1%), and minor product C (1.5 mg, 7-..6%).
Each purified component was characterized by spectroscopic analysis. Compound A was identified as 7,8-cis-1a,25-dihydroxyvitamin D3 (6, analog JR), compound B as the starting vinylallenol JV (4) and compound C as 1a,25-dihydroxy-cis-isotachysterol.
1H~NM$ (300 I~iZ) : (CDC13) a 0. 64 (3H, C1$-CH3, s) , 0.95 {3H, C21-CH3, d, J'6.4 Hz) , 1.22 (6H, CZS,z~-2CH3, s) , 1.0-2.1 to (remaining ring and side chain hydrogens, series of m), 2.24 (1H, dd, J'12.4 Hz, 9.0 Hz), 2.55 {iH, dd, J'12.5 Hz, 3.4 Hz), 4.17 (1H, C3-H, dddd,J'4.2 Hz, 4.2 Hz, 4:2 Hz, 4.2 Hz), 4.42 (iH, C1-H, br s) , 5.01 (iH, C19-H, br s) , 5.32 (iH, C19-H, br s), 6.20 and 6.54 (2H, C6-H and C~-H, AB pattern, J'11.5 Hz).
13~ (75.5 MHZ): (CDC13) b 12.6, 19.1, 20.9, 24.1, 26.3, 28.4 , 29.2, 29.4, 36.1, 36.5, 39.0, 40.7, 42.7, 44.4, 45.9, 46.7, 55.0, 56.1, 66.6, 71.1 , 72.1, 113.9, 121.2, 126.2, 133.1, 142.5, 146.3.
uy: (100% EtOH) 1~x 266 nm (E 15, 000) ; l~"in 228 nm (e _ 20 9,300) . Hue: m/z 416.3281 (calcd. for C2~H4,O3,- 416.3292) .
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).

~'~~ica~ Sxnthesis of Analog JS
This example illustrates preparation of analog JS, namely 5,6-traps-7,8-cis-1a,25-dihydroxyvitamin D3, as seen in Scheme H.
Preparation of sulfur dioxide adducts A and B of 7,8-cis-1a,25-dihydroxyvitamin D3, compounds (7a) and (7b).
A solution of the 7,8-cis-isomer 6 (15.6 mg, 0.0374 mmol) in dichloromethane (4 mL) was cooled to -15°C. Sulfur dioxide (5 mL), pre-dried by passage through concentrated sulfuric acid, was condensed into the cooled reaction flask. The solution was stirred for 3 h at -15°C and then the mixture was slowly warmed to room temperature, allowing the SOZ to boil off. The solvent was removed under reduced pressure and pure product was obtained by HPLC (100% ethyl acetate, Rainin Microsorb column, 4 mL/min flow rate) as two fractions, A (7.2 mg, 40%; colorless, solid residue) and B (5.5 mg, 31%;
colorless, solid residue). A and B were identified as the two epimeric sulfone adducts 7a and 7b, but absolute stereochemical identification was not attempted.
Spectral Data for Adduct A (7a):
1F;~NM$ (300 MHZ) : (CDC13) b 0.68 (3H, C18-CH3, s) , 0.96 (3H, C21-CH3, d, J'6.2 Hz) , 1.22 (6H, C2s,2~-2CH3, s) , 1.25-2.38 (remaining ring and side chain hydrogens, series of m), 3.68 (1H, C19-H, d, J'16.2 Hz), 3.98 (IH, C19-H, d, J'16.2 Hz), 4.24 (iH, C3-H, dddd, J'4.3 Hz, 4.3 Hz, 4.3 Hz, 4.3 Hz), 4.40 (iH, C1-H, br s) , 4.93 and 5.02 (2H, Cs-H and C~-H, AB pattern, J'i1.2 Hz).
(75.5 MHZ): (CDC13) b 12.9, 19.1, 20.9, 23.8, 26.5, 28.1, 29.3, 34.4, 36.2, 36.4, 39.0, 40.2, 40.4, 44.3, 46.2, 55.0 ,55.1, 55.8, 63.8, 65.5, 66.9, 71.1, 111.8, 128.8, 134.0, 150.6.
Ig: (CC14) v 3200-3600 (C-OH, br s), 2880-2980 (C-H, s), 1660-1680 (C=C, w) , 1315 (sulfone, s) , 1115 (sulfone, m) cni 1.
HBM~: FAB (NBA) , m/z 479.2849 (calcd. for C~Hq4O5S minus H, 479.2833).
Spectral data for Adduct B (7b):
leg (30o MHz) : (cncl,) a o.73 (3H, C18-CH3, s) , 0.95 (3H, CZi-CH3, d, J'6.4 Hz) , 1.21 (6H, CZS,2~-2CH3, s) , 1.25-2.09 (remaining ring and side chain hydrogens, series of m), 2.29 (iH, br d, J'13.1 Hz), 2.46 (iH, br d, J'17.5 Hz), 3.70 (iH, C19-H, d, J'15.8 Hz), 4.01 (1H, C19-H, d, J'15.8 Hz), 4.23 (1H, C3-H, m) , 4.40 (1H, C1-H, br s) , 4.87 and 4.98 (2H, C6-H and C~-H, AB pattern, J'i1.0 Hz).
13~-NMB (75.5 MHZ): (CDC13) b 12.7, 19.1, 20.9, 23.9, 25.8, 28.4, 29.1, 29.4, 33.8, 35.9, 36.5, 39.1, 40.0, 40.6, 44.3, 46.9, 55.0, 55.3, 55.7, 64.0, 65.0, 66.9, 71.2, 112.4, 128.6, 134.0, 150.8.
fig: (CC14) v 3200-3600 (C-OH, br s), 2860-2980 {C-H, s), 1650-1680 (C=C, w) , 1315 (sulfone, s) , 1115 (sulfone, m) cm 1.
FAB (NBA) , m/z 479.2822 (calcd. for Cz,H9405s minus H, 479.2833).
Preparation of 5,6-traps-?,8-cis-1a,25-dihydroxyvitamin D3 (8, Analog JS) via Sulfur Dioxide Adducts The sulfone Isomer A (7a, 4.0 mg, 0.0083 mmol) and NaHC03 (14 mg) were dissolved in ethanol (5 mL). The solution was flushed with argon for 10 min, then heated at 78°C for 1.5 h.
Solvent was removed and the crude product, obtained by f lash chromatography (silica gel, 80% ethyl acetate/hexanes), was subjected to HPLC purification (80% ethyl acetate/hexanes, Rainin Microsorb column, 4 mL/min flow rate) to afford pure 5,6-trans-7,8-cis-1a,25-dihydroxyvitamin D3 (3.3 mg, 95%) as a colorless, viscous foam. Likewise, treatment of sulfone Isomer B (7b, 3.3 mg, 0.0069 mmol) with NaHC03 (15 mg) in ethanol ( 5 mL) followed by work up and purification exactly as above afforded pure 8 (2.5 mg, 86%) as a colorless, viscous foam .
Spectral data:
1Fi-NMg (300 MHZ) : (CDC13) a 0.66 (3H, Cle-CH3, s) , 0.96 (3H, C21-CH3, d. J'6.3 Hz) , 1.22 (6H, 026,2-,-2CH3, s) , 1.24-2.34 (remaining ring and side chain hydrogens, series of m), 2.78 (iH, dd, J'12.9 Hz, 2.7 Hz), 4.20-4.28 (iH, C3-H, m, W'26 Hz), 4.45-4.52 (1H, C1-H, m, W'23 Hz), 4.95 (1H, C19-H,-br s), 5~05 (iH, Cls-H, br s) , 6.15 and 6.75 (2H, C6-H and C~-H, AB
pattern, d, J'11.8 Hz).
13C-NMB (75.5 MHZ): (CDC13) a 12.7, 19.1, 20.9, 24.2, 26.4, 28.4, 29.2, 29.4, 29.7, 35.9, 36.1, 36.5, 39.4, 40.7, 42.0, 44.4, 46.8, 55.0, 56.2, 66.0, 70.9, 109.1, 120.1, 124.6, 133.1, 144.2, 152Ø
jJy: (100% EtOH) 1~",ax 274 nm (E 17,400) ~ ?~",in 234 nm (E'.
5, 500) .
m/z 416.3284 (calcd. for C2-,H44O3, 416.3292) .
~.: m/z 416 (15, M) , 398 (12) , 380 (10) , 365 (4) , 342 (3) , 329 (2) , 313 (3) , 287 (7) , 269 (7) , 251 (9) , 227 (5) , 209 (6), 175 (12), 152 (28), 134 (base), 107 (22), 95 (30), 81 (29), 69 (30), 59 (42).
Ex~MgT.F 1 ~
~hem~cal Synthes~s of Ana~o~r JV
This example illustrates preparation of JV, namely (1S,3R,6S)-1,3,25-trihydroxy-9,10-secocholesta-5(10),6,7-triene as seen in Scheme H.

Preparation(1S,3R,8S)-8-benzoyloxy-1,3-di[(tert-butyldimethylsilyl)oxy]-25-trimethylsilyloxy-9,10-secocholest-5(10)-en-6-yne (3).
To A-ring enyne 1 (483 mg, 1.36 mmol) in dry ether (1.6 mL) under an argon atmosphere at 0°C was added n-BuLi (1.4 mmol, 0.88 mL, 1.6 M in hexanes). The solution was stirred for 1 h at 0°C, then the ketone 2 (402 mg, 1.14 mmol) in ether (3 mL) was added dropwise. The solution was stirred at 0°C
for 10 min, then warmed to room temperature. After stirring the mixture for 1 h, brine (1 mL) was added, the mixture was diluted with ether (10 mL), and the aqueous layer was extracted with ether (2 x 10 mL). The combined ether extracts were dried (MgS04) . The residual oil after evaporation was purified by flash chromatography (silica gel, 5% ethyl acetate/hexanes) followed by HPLC (5% ethyl acetate/hexanes, Rainin Dynamax colwan, 8 mL/min flow rate) to afford pure product(1S,3R,8S)-8-Hydroxy-1,3-di(tert-butyldimethylsilyloxy)-25-trimethylsilyloxy-9,10-secocholest-5(l0)-en-6-yne (661 mg, 79% yield). The propargyl alcohol was identified by spectroscopic analysis. -(300 MHZ): (CDC13) b 0.06 (6H, Si-2CH3, s), 0.09 (6H, Si-2CH3, s), 0.10 (9H, Si-3CHj, s), 0.9-1.0 (24H, series of overlapping signals due to 2 Si-tBu, Cle-CH3 and C21-CHj} , 1.20 (6H, C26,27-~3~ s) ~ 1.87 (3H, Cl9-CH3, br s) , 0.97-2.39 (remaining ring and side chain hydrogens, series of m), 4.03-4 .12 ( 1H, C1-H, m, W-26. 7 Hz ) , 4 .17 ( 1H, C3-H, br s) .
1'c-NMIt ( 7 5 , 5 MHZ ) : ( CDC13 ) b -4 . 8 , -4 . 7 , =4 . 6 , -4 . 3 , 2.6, 13.0, 18.0, 18.1, 18.4, 18.6, 19.1, 20.8, 21.1, 25.8, 25.9, 26.7, 29.8, 29.9, 35.3, 36.2, 39.7, 40.0, 40.4, 41.2, 42.5, 45.2, 56.3, 56.9, 64.1, 69.8, 69.9, 74.1, 82.1, 96.6, 114.7, 141.3.
(FAB) m/z 731.5295 (calcd. for C~ZH8004S13, 733.318).
m/z 731 (5, M-H}, 715 (11, M-OH), 676 (2), 625 (2}, 600 (21) , 583 (12) , 569 (3) , 493 (3) , 469 (3) , 437 (4) , 379 (6) , 355 (5) , 323 (5} , 301 (7) , 275 (8) , 249 (18) , 223 (9) , 191 {11), 165 (25), 157 (10}, 147 {54), 131 (base).
To the propargyl alcohol (586 mg, 0.818 mmol} in dry ether (3 mL) at -78°C under an argon atmosphere was added n-BuLi (0.88 mmol, 0.55 mL, 1.6 ~ in hexanes). The solution was warmed to room temperature and stirred for 2.3 h then recooled to -78°C. Freshly distilled benzoyl chloride (103 ,uL, 0.883 mmol) was added dropwise. The solution was warmed to room temperature and stirred for 2 h. The reaction was quenched with saturated aqueous NaHCO3 (1 mL) and diluted with ether (20 mL). The organic layer was washed with NaHC03 (2 x 5 mL) and brine (1 x 5 mL) and dried (MgS04). 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.
~ (300 MHZ): (CDC13) b 0.05 (6H, Si-2CH3, s), 0.08 (6H, Si-2CH3, s), 0.11 (9H, Si-3CH3, s), 0.87 (9H, Si-tBu, s), 0.88 (9H, Si-tBu, s), 0.93 (3H, CZ1-CH3, d, J'6.5 Hz), 1.04 (3H, C18-CH3, s) , 1.21 (6H, ~6,27-CH3, s) , 1.88 (3H, C19-CH3, s) , 1.26-2.08 (remaining ring and side chain hydrogens, series of m), 2.36 (iH, dd, J'16.7 Hz, 4.5 Hz), 3.12 (1H, d, J'10.1 Hz), 4.01-4.09 (1H, C3-H, m, W'32 Hz), 4.14 (1H, C1-H, br s), 7.43 (2H, m-Ar, t, J'7.4 Hz, 7.7 Hz), 7.55 (1H, p-Ar, t, J'7.3 Hz), 8.05 (2H, o-Ar, d, J'7.4 Hz).
1'CNMRNMR (75.5 MHZ): (CDC13) b -4.8 , -4.7 , -4.6 , -4.3, 2.7, 13.9, 18.0, 18.1, 18.5, 18.7, 19.1, 20.8, 21.4, 25.8, 25.9, 26.6, 29.9, 30.0, 35.4, 35.8, 36.1, 39.5, 39.7, 41.3, 42.6, 45.2, 57.0, 57.5, 64.1, 64.9, 74.1, 77.1, 84.5, 92.1, 114.8, 128.3, 129.6, 131.5, 132.6, 141.8, 164.5.
=$: (CC14) v 3590 (monosubstituted benzene, w), 2870-2980 (C-H, S) , 2220 (C_C, w) , 1745 (C=O, S) cm~l.
(100% EtOH) ?~,uax 232 nm (E 23,700) .
(FAB) m/z 835.5564 (calcd. for C49He40sSis minus H, 835.5551).
~: 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).
Preparation of analog JV, (1S,3R,6S)-1,3,25-trihydroxy-9,10-secocholesta-5(10),6,7-triene (4) Freshly purified 1,2-diiodoethane (412 mg, 1.46 mmol) and samarium metal (286 mg, 1.90 mmol) were dried under vacuum and suspended in 4 mL THF under an argon atmosphere. This solution was stirred for 2 h until it became deep blue. A
solution of propargyl benzoate 3 (477 mg, 0.570 mmol) and Pd(PPh3)4 (65.8 mg, 0.037 mmol) in 6 mL THF was added via cannula. Freshly distilled isopropanol (from CaO, 0.5 mL) was added and the solution was stirred under a positive argon atmosphere for 14 h. Saturated aqueous Na2C03 (2 mL) was added to quench the reaction. The organic layer was diluted with ether and then the mixture was washed with Na2C03 (3 x 10 mL), dried with MgS04 and concentrated. 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 (iS,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 1H-NMR analysis and immediately deprotected as described below. This material appeared to be more stable as the triol 4.
Spectral data:
(300 MHZ): (CDC13) b 0.06 (6H, Si-2CH3, s), 0.10 (9H, Si-3CH3, s) , 0.11 (6H, Si-2CH3, s) , 0.73 (3H, C21-CH3, s) , 0.89 (9H, Si-tBu, s), 0.91 (9H, Si-tBu, s), 0.94 (3H, Cle-CH3, d, J-6. 5 Hz) , 1.20 (6H, C26,2~-CH3, s) , 1.76 (3H, C19-CH3, s) , 1.26-2.50 (remaining ring and side chain hydrogens, series of m), 4.09-4.13 (iH, C3-H, m, overlapping C1-H), 4.17 (iH, C1-H, br distorted singlet), 6.13 (1H, C6-H, dd, J-3.9 Hz, 3.9 Hz).
Minor impurity peaks were detectable and this compound was best characterized as the deprotected triol.
To the silyl protected vinylallene (0.1054 g, 0.1469 mmol) was added tetra-n-butyl ammonium fluoride (1 M in THF, 1.6 mL, 1.6 mmol). The solution was stirred under an argon atmosphere for 19 h. Water (1 mL) was added and the solution stirred 30~min. The mixture was extracted with ether (3 x 15 mL) and the ether extracts washed with brine (1 x 10 mL) and dried (MgS04). The concentrated residue was subjected to WO 99/16452 PCTlUS98119862 f lash chromatography (silica gel, 80% ethyl acetate/hexanes) followed by HPLC (80% ethyl acetate/hexanes, Rainin Microsorb column, 4 mL/min flow rate) to afford purified deprotected vinylallene 4 (Analog JV) together with its 6R-diastereomer 5 (Analog JW) (46.1 mg, 75.3% total yield) in a -92:8 ratio by NMR integration. By a tedious HPLC separation (same conditions as above by shave-recycling), pure 5 could be obtained and characterized by spectroscopic analysis:
The data for compound 4 are as follows:
l~irNM$ (300 MHZ) : (CDC13) a 0.74 (3H, C18-CH3, s) , 0.95 ( 3H, C21-CH3, d, J-6. 4 Hz) , 1. 22 ( 6H, Cy6,27-CH3 i s) . 1. 87 (3H, Cl9-CH3, s), 1.25-2.10 (remaining ring and side chain hydrogens, series of m), 2.29 (iH, br d, J-13.2 Hz), 2.62 (iH, br dd, J"16.5 Hz, 4.5 Hz), 4.11-4.20 (1H, C3-H, m, W"27.8 Hz), 4.23 (1H, Cl-H, br m W-8.6 Hz), 6.14 (1H, C6-H, dd, J'4.1 Hz, 4.1 Hz).
uy: (100% EtOH) lax 242 nm (E 24,300), 234 nm (e 23,500) .
m/z 416.3277 (calcd. for CZ~Hq9O3, 416.3292) .
M~: 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) .
a ~'hcal Synthesis of Analog JW
This example illustrates preparation of the analog JW, namely,(1S,3R,6R)-1,3,25-trihydroxy-9,10-secocholesta-5(10),6,7-triene (5), as seen in Scheme H.
A solution of (6S/6R)-vinylallenes 4, 5 (2.6 mg, 0.0062 mmol, an -92:8 ratio of 6S:6R) in methanol-d4 (1 mL) was prepared in a quartz NMit tube. The solution was saturated with argon for 30 min and then the NMFt tube was capped and then irradiated with ultraviolet light from a Hanovia 450 watt medium pressure mercury lamp for 30 min. Integration of the C18-Me signals in the NMIt spectrum revealed a "50:50 mixture of the two isomers. Solvent was removed and the products separated by HPLC (11% isopropanol/hexanes, Rainin Microsorb column, 6 mL/min, flow rate). Taking a front cut of the overlapping peaks gave pure (6R)-vinylallene 5 (0.9 mg, 35%).
This product was identified and characterized through spectroscopic analysis.
1~-NMg (300 MHZ) : (CDC13) b 0.65 (3H, Clg-CH3, s) , 0.94 (3H, C21-CH3, d, J-6.4 Hz) , 1.21 (6H, C26,2~-2CH3, s) , 1.87 (3H, C19-CH3, br s), 1.25-2.32 (remaining ring and side chain hydrogens, series of m), 2.28 (1H, br d, J-13.0 Hz), 2.52 (1H, dd, J-16.3 Hz, 5.0 Hz), 4.12 (1H, C~-H, m, W-30.0 Hz, overlapping), 4.20 (1H, C1-H, br s), 6.10 (iH, C6-H, dd, J'3.2 Hz, 3.2 Hz) .
jly: ( 100% EtOH) Jv""X 242 not (e 22, 300) , 234 nm (e 22,100).
m/z 416.3291 {calcd. for C2~H4~O3, 416.3292) .
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 described in Examples 21 and 22.
Scheme I
PhpP=O
t) n-BuLi or PhLi, -78 °C, THF
1 ~ 2) TBDMSO' ~ ~ OTBDMS ~ 3b~ met 2a, para o H 2b, meta TeDMSO' 4) TBAF, THF
3) TBAF, THF '~a 3B ' via 3a .'~ ~..n H 4 ~ H
Analog JX I ~ Analog JY
HO'-Chemi cal synthesis of Ana,~g JX -This example illustrates preparation of the analog JX, namely 22-(p-hydroxyphenyl)-23,24,25,26,27-pentanor-vitamin D3 (4), as seen in Scheme I.
The A-ring phosphine 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) Was added dropwise 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 stirred 2 h at -78°C, warmed to room temperature and quenched with water (5 mL). The aqueous layer was separated and extracted with EtOAc (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2S04, and concentrated. The crude residue was purified by rapid filtration through a short silica 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). After extracting the aqueous layer with EtOAc (3 x 5 mL), the combined organic layers were washed with brine and dried over Na2S0,. The residue was then purified by HPLC
(20% EtOAc/hexanes) to afford, after vacuum drying, 4.7 mg (36%) of the desired product 4 (Analog JX).
'~ (300 MHZ) : (CDC13) b 0.57 (3H, C18-Me) , 0.81 {3H, H21, d, J-6.4 Hz), 1.2-1.5 (remaining ring and side chain hydrogens, series of m), 2.58 (dd, J-13.0 Hz, 3.0 Hz), 2.83 (dd, J"13.1 Hz, 3.0 Hz), 3.96 (1H, H3, m), 4.83 (iH, H19, br s), 5.06 (1H, H19., br s), 6.05 (iH, d, J-11.2 Hz), 6.24 (1H, d, J-11.2 Hz), 6.74 (2H, Ar-H3.,5., d, J"8.4 Hz), 7.00 (2H, Ar-H2, 6, d, J" 8 . 3 Hz ) .
jJy: (100% EtOH) 1~",ax 266 nm (E 20, 600) ; ?~" 240 nm (E
15, 000) .
m/z 406.2855 (calcd. for C2BH38O2, 406.2873) .
m/z 406 (23, M) , 388 (3) , 373 (11) , 347 (35) , 299 (4), 281 (5), 253 (45), 239 (3), 211 (5), 197 (5), 158 (14), 13 6 { 29, A-ring fragment due to C-,,e cleavage) , 118 ( 30, - m/ z 136-H20), 107 (base), 91 (20), 81 (16), 67 (10), 55 (17).
RxAMpLE 22 Chem~ca~ $yrthe~~s of Analog JY
This example illustrates preparation of the analog JY, namely 22-(m-Hydroxyphenyl)-23,24,25,26,27-pentanor-vitamin D3 (5), as seen in Scheme I.
The A-ring phosphine oxide 1, (70 mg, 0.154 mmol) in dry THF (2.8 mL) was cooled to -78°C under argon and n butyllithium (1.5 M in hexanes, 0.100 mL, 0.154 mmol) was added via a syringe. 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 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 Na2S04 and concentrated.
The crude residue was purified by rapid filtration through a short silica gel column {20% EtOAc/hexanes) to yield 19.2 mg (29%) of the protected vitamin 3b. The protected vitamin (19.2 mg, 0.03 mmol) in dry THF (1 mL) was placed under argon and TBAF (1 M in THF, 0.30 mn, 0.30 mmol) was added dropwise.
After stirring 18 h, the solvent was partially evaporated and diluted with water (5 mL). After extracting the aqueous layer With EtOAc (3 x 5 mL), the combined organic layers were washed with brine and dried over Na2S04. The residue was purified by HPLC (20% EtOAc/hexanes) and after vacuum drying afforded 2.8 mg (23%) of the desired product 5 (Analog JY).
1H-NMR (300 MHZ) : (CDC13) S 0.58 (3H, Hle-CH3, s) , 0.83 ( 3H, HZO-CH3, d, J-6.5 Hz) , 1.2-1. 5 (remaining ring and side chain hydrogens, series of m), 2.58 (1H, dd, J'13.0 Hz, 3.3 Hz) , 2. 85 (2H, H22, m) , 3.97 (1H, H3, m) , 4.83 (1H, H19, s) , 5.07 (1H, H19~, s) , 6.06 (iH, H6,~, AB pattern, d, J'll. 2 Hz) , 6.24 {1H, H6,~, AB pattern, d, J'11.2 Hz), 6.63 (1H, Ar H, s), 6.64 (1H, Ar H, d, J-7.4 Hz), 6.71 (1H, Ar H,,d, J-7.52 Hz), 7.13 (iH, Ar H, dd, J'15.45 Hz, 7.8 Hz).

$$~j$,: m/z 406.2872 (calcd. for C28H38~2~ 406.2873) .
m/z 406 (44) , 373 (14) , 347 (7) , 299 (6) , 271 (9) , 253 (7) , 2Il (12) , 176 (20) , 158 (30) , 136 (23) , 118 (54) , 107 (35) , 91 (23) , 79 (22) , 67 (12) , 55 (11) .
Scheme J relates to analog LO described in Example 23.
y cheme J
or. ~ off 2 O 3 3) T~.1S-Imidazote v ~OTMS
I~ 4 a) ~o~ T
_ O
~OTMS
v ~OTMS

.
OTf "
Pd(PPh~)2(OAc) . ~ ~ 7 61 TBAF, TBDMSO"" OTBDMS Cul, EtpNH. oM~, rt mF

TBDMSO' - v ~OTBDMS HO-"
/ 7) Hz, Pd, lindla~
~OH r quinotine, hexanes v ~OH
" 8) acetone, heat OH
~ ~o, Analog LO
HO

This example illustrates preparation of the analog LO, namely (14R,15S)-14,15-methano-1a,25-dihydroxyvitamin D3 (10)as seen in Scheme J.
Preparation of (8R,14R,15S)-de-A,B-14(15) cyclopropylcholest-8-of (2). Into a dry 250 mL Schlenk tube flushed with argon and equipped with a stir bar was placed the (8R)-De-A,B-cholest-14-en-8-of (1)(1.50 g,5.6 mmol), diiodomethane (15.0 g, 4.5 mL, 56 mmol) and dry CH2C12 (100 suBSmur~ sHeEr ~au~ 2s~
j oh t ) CHZty 1 EtZZn 2y RuOa 1 CH3CN-CCI, I "

mL). The mixture was cooled to -78°C while stirring. Diethyl zinc (1.0 M solution in hexanes, 28.0 mL, 28 mmol) was added to the mixture via gas tight syringe. The mixture was stirred at -78°C for 4 h and then allowed to warm to room temperature overnight. The mixture was then treated with saturated NH9C1 and extracted with ether (3 x 50 mL). The combined ethereal phase was washed with saturated NaHC03 and brine and dried over MgSO,. The solvent was removed to give a yellow milky liquid. Flash chromatography (20% EtOAc/hexanes) afforded 2 as a thick, colorless oil (1.24 g, 79%).
(300 MHZ, CDC13): S 0.23 (dd, J'3.9, 2.8 Hz, iH, He) , 0.39 (dd, J'7.7, 4. 3 HZ, 1H, Ha) , 0.80-0.90 (m, 12H, C18 Me, C21-Me, C26,2~-2Me) , 0.90-2.00 (remaining ring and side chain hydrogens, series of m), and 4.16 (dd, J'10.8, 4.2 Hz, 1H, H~ ) .
13s-NMB (75.5 MHZ, CDC13): 5 5.1, 15.3, 17.6, 18.7, 21.7, 22.5 , 22.8, 23.7, 28.0, 32.4, 33.8, 35.0, 35.5, 36.1, 39.5, 40.9, 43.2, 49.0, and 66.8. ~,$ (CC14): v 3320 (O-H) and 2940 (C-H).
M~. (m/z) : 278 (M+, 12%) , 261 {M+-OH, 23) , -260 (M+-H20, 14) , 175 (16) , 165 (M+-CeHl~, 29) , 149 (12) , 148 (17) , 147 (89), 123 (10), 121 (14), 111 (base), 109 (12), 105 (15), 95 (18), 93 (11), 91 (13), 81 (16), 57 (12), 55 (14), and 43 (26) .
FYac-t Mass (m/z) : calculated for C19H39O: 278.2610.
Found: 278.2608.
Preparation of (14R,15S)-de-A,B-14(15)-cyclopropyl-25-hydroxycholest-8-one (3) Into a 10o mL round bottom flask was placed the a cyclopropyl alcohol 2 (1.21 g, 4.52 mmol), NaI04 (3.38 g, 15.8 mmol) , RuCI3~XH20 (0.187 g, 0.90 mmol) and a stir bar. The mixture was dissolved in CH3CN (18.1 mL), CC14 (18.1 mL) and 0 . 5 M IOi2P04 + 0 . 5 M NaOH ( 22 . 6 mL) . The mixture was degassed and flushed with argon. The mixture was stirred at 54 °C.
After 10 min the mixture turned from black to yellow. After 18 h, the solution turned black. The mixture was treated with brine and extracted several times with ether. The ether layer was dried over MgSO9 filtered and concentrated. The crude pCT/US98119862 could be flushed with 20% EtOAc/hexanes but was purified via HPLC (Rainin Dynamax-60A, 2.14 x 25 cm, 8~m silica gel column, 25% EtOAc/hexanes, 8 mL/min) to afford 3 as a colorless oil (0.332 g, 25% yield). IHrNMB (300 MHZ, CDC13): b 0.31 (dd, J-8. 0, 4. 0 HZ, 1H, Ha) , 0.80 (S, 3H, C18-Me) , 0.86 (d, J-6.4 Hz, 3H, C21-Me), 0.90-2.36 (remaining ring and side chain hydrogens, series of m) , and 1.14 (s, 6H, CZ-,,ZS-2Me) .
isC-NMBNMB (75,5 MHZ, CDC13): b 18.4, 18.6, 18.7, 19.4, 20.6, 21.4, 29.2, 29.3, 31.5, 33.7, 34.4, 36.0, 38.4, 42.7, 44.2, 46.9, 47.9, 70.8, and 211.9.
_$ (CC14): v 3448 (O-H), 2966 (C-H), and 1701 (C=O).
uy (loo% EtoH) : ~",ax 212 nm (E 1400) .
(m/z) : 292 (M+, 1.3%) , 274 (M~-HZO, 13) , 164 (25) , 163 (36) , 150 (12) , 149 (19) , 147 (14) , 145 (18) , 137 (25) , 136 (71), 135 (37), 136 (71), 137 (25), 105 (22), 95 (18), 93 (25) , 92 (13) , 91 (43) ,. 81 (17) , 79 (34) , 77 (21) , 69 (22) .
67 (22), 61 (43), 59 (59), 55 (38), 45 (35) , 44 (19), and 43 (base).
Exact Mass (m/z) : calculated for C19H32O2: 292.2402.
Found: 292.2407.
Preparation of (14R,15S)-de-A,B-25-trimethylsilyloxy-14(15)-cyclopropylcholest-8-one (4) Into a dry 25 mL round bottom flask equipped with a stir bar and flushed with argon was placed the 25 hydroxycyclopropylketone 3 (0.320 g, 1.09 mmol) and dry THE
(14 mL). TMS-imidazol (0.48 mL, 3.27 mmol) was added via syringe and the mixture was allowed to react overnight.
Afterwards, the reaction mixture was immediately flushed through a short silica gel column (10% EtOAc). HPLC (Rainin Dynamax-60A, 2.14 x 25 cm, 8~m silica gel column, 10%
EtOAc/hexanes, 8 mL/min) afforded 4 as a colorless oil (0.327 9. 82%).
1~ (300 MHZ, CDC13): S 0.06 (s, 9H, SiMe~), 0.33 (dd, J"8.0, 4.0 Hz, iH, Ha) , 0.83 (s, 3H, C18-Me) , 0.88 (d, J'6.5 Hz, 3H, C21-Me), 0.93-2.38 (remaining ring and side chain hydrogens, series of m) , and 1.16 (s, 6H, CZS,2~-2Me) .
isC-NMBNMB (75,5 MHZ, CDC13): 5 2.6, 18.4, 18.6, 18.8, 19.4, 20.6, 21.5, 29.8, 30.0, 31.5, 33.8, 34.5, 36.0, 38.5, 42.7, 45.1, 46.9, 47.9, 74.0, and 211.8.
~$ (CC14) : v 2956 (C-H) and 1707 (C=O) .
jay (100% EtOH) : 1~",ax 218 nm (e 2000) . ~ (m/z) : 365 (MH+, 5%) , 349 (19) , 275 (30) , 163 (39) , 135 (12) , 132 (13) , 131 (base), 91 (13), 75 (42), 73 (41), 69 (12), 59 (18), 55 (16), and 43 {27).
Exact Mass (m/z) : calculated for Cz2H410ZSi (MH') 365.2876. Found: 365.2867.
Preparation of (14R,15S)-de-A,B-25-trimethylsilyloxy 14(15)-cyclopropylcholest-8-en-8-yl trifluoromethane sulfonate (5) .
Lithium di-isopropyl amide (LDA) was prepared by the addition of di-isopropyl amine (0.097, 0.69 mmol) to a solution of n-BuLi in hexanes (0.48 mL, 1.6 M, 0.77 mmol) and dry THE (1 mL) at -78°C. After stirring for 10 min at -78°C
and at room temperature for 15 min the solution was again cooled to -78°C and the 25-TMS cyclopropylketone 4 (0.200 g, 0.548 mmol) in THE (2 mL) was added dropwise via a cannula.
After stirring for 15 min the enolate solution was warmed to room temperature over 2 h and then cooled to -78 °C. N-phenyl trifluoramide (0.218 g, 0.61 mmol) was dissolved in dry THE
(2 mL), and added to the enolate at -78 °C. The reaction mixture was warmed to 0 °C and stirred for 10 h. The resulting solution was poured into Water and extracted with ether, dried over MgS04, and concentrated. The yellow solid was chromatographed (hexanes) to afford 5 as a colorless oil (0.163 g, 63%).
(300 MHZ, CDC13): b 0.10 (s, 9H, SiMe3), 0.58 (dd, J"7 .8, 4.7 Hz, 1H, H,) , 0.73 {apparent t, J-4 . 0 HZ, 1H, Hb) , 0.90 (d, J-6.5 Hz, 3H, C21-Me), 0.98 (s, 3H, Cle-Me), 1.00-2.50 (remaining ring and side chain hydrogens, series of m), 1.19 (s, 6H, CZS,z?-2Me) , and 5.56 (t, J-3.7 Hz, iH, H9) .
1~C~NMR (75.5 MHZ, CDC13): S 2.6, 14.2, 15.1, 18.7, 20.6, 21.3, 23.7, 29.8, 30.0, 31.8, 32.8, 34.1, 36.2, 37.1, 43.4, 45.1, 46.7, 74.0, 114.7, and 150.2.
Z$ (CC14): v 2958 (C-H) and 1420, 1248 (S=O).
jay (100% EtOH) : 1,",aX 216 nm (e 3700) .
(m/z) : 495 (MH+, 3%) , 147 (17) , 145 (18) , 143 (14) , 133 (14), 132 (13), I31 (base), 129 (12), 119 (11), 117 (13), 115 (21) , and 105 (18) .
Exact Mass (m/z) : calculated for C23Hse04F3SSi (MH+) 495.2212. Found: 495.2234.
5 Preparation of (iS,14R,15S)-1,3-di(tert-butyldimethylsilyloxy}-25-trimethylsilyloxy-14(15)-cyclopropyl-6,7-dehydroprevitamin D3 (7) To a mixture of enol triflate 5 (76.9 mg, 0.155 mmol) and enyne 6 (65 mg, 0.171 mmol) in diethylamine (1 mL) and DMF (1 10 mL) was added CuI (3mg, 0.0155 mmol) and bis[triphenylphosphine]palladium (II} acetate (3.5 mg, 0.0047 mmol). The reaction mixture was stirred at room temperature for 2 h under argon. Diethyl ether was added, and the mixture was washed with water (3 x 5 mL), dried (MgSO,}, and 15 evaporated in vacuo. The crude dark brown oil was purified by flash chromatography (5% EtOAc/hexane) to afford after vacuum drying 109.6 mg (97%) of the dienyne 7 as a viscous oil, which was sufficiently pure for the next step.
(300 MHZ, CDC13} : b 0.06 (s, 6H, SiMe2) , 0. 09 (s, 20 6H, SiMe2}, 0.10 (s, 9H, SiMe3), 0.46 (dd, J-7.5,-4.0 Hz, 1H, Ha) , 0.59 (apparent t, J"3.6 Hz, 1H, Hb) , 0.88 (s, 9H, SitBu) , 0. 89 (s, 9H, SitBu) , 0.90 (superimposed signal, 3H, C21-Me) , 0.92 (s, 3H, Cle-Me), 0.94-2.43 (remaining ring and side chain hydrogens, series of m), 1.19 (s, 6H, C26,2~-2Me), 1.86 (br s, 25 3H, C19-Me) , 4. O8 (m, iH, H3) , 4.18 (apparent t, J"3 . 2 Hz, 1H, Hl}, and 5.95 (apparent t, J'3.8 Hz, 1H, H9).
(75.5 MHZ, CDC13): a -4.8, -4.7, -4.65, -4.4, 2.6, 14.9, 15.1, 18.0, 18.7, 19.1, 20.6, 20.7, 25.2, 25.8, 25.9, 29.8, 29.9, 32.5, 32.7, 34.3, 36.2, 37.9, 39.8, 40.5, 30 41.2, 45.2, 47.2, 64.1, 69.9, 74.0, 87.9, 90.2, 115.3, 125.2, 132.1, and 140.4.
(NaCl): v 2970, 2880 (C-H), 2190 (C_C), and 1615 tC=C) .
(DCI, NH3): m/z 727 (MH;, 5%), 596 (23), 595 (26), 594 35 (30), 147 (11), 132 (10), 131 (67), 92 (15), 91 (19), 90 (14), 76 (13), 75 (base), 74 (33), 73 (33), 58 (10), 56 (12), and 43 (10) .
Exact Mass (DCI, NH3/PEG) : calculated for C43H79O3Si3 (MH*): m/z 727.5337. Found: m/z 727.5345.
Preparation of (1S,14R,15S)-1a,25-dihydroxy-14(15)-cyclopropyl-6,7-dehydroprevitamin D3 (8) To a solution of dieyne 7 (109.6 mg, 0.1507 mmol) in 5 mL of THF under argon was added tetrabutylammonium fluoride (1.13 mL, 1 M in THF, 1.13 mmol). The reaction mixture was stirred at room temperature in the dark for 12 h. It was diluted with ethyl acetate and washed with brine (2 x 1o mL).
The aqueous layer was extracted with ethyl acetate (2 x 10 mL), and the combined organic layer was dried (MgSO,) and evaporated in vacuo. Flash chromatography of the residual oil (100% EtOAc) afforded after vacuum drying 59.6 mg (93%) of the triol 8 as a colorless oil, which was sufficiently pure for characterization and further reaction.
1~ (300 MHZ, CDC13): b 0.45 (dd, J-7.6, 4.3 Hz, 1H, Ha), 0.60 (apparent t, J-3.7 Hz, 1H, I~ih), 0.85-2.60 (remaining ring and side chain hydrogens, series of m), 0.90 (d, J-6.6 Hz, 3H, C21-Me) , 0.92 (s, 3H, C18-Me) , 1.21 (s, 6H, CZS,2,-2Me) , 1. 97 (br s, 3H, Ci9-Me) , 4.11 (m, 1H, H3) , 4.25 (apparent t, J-3.9 Hz, iH, Hl), and 5.98 (apparent t, J-3.8 Hz, iH, H9).
13C-NMR (75.5 MHZ, CDC13): 5 15.0, 15.2, 18.7, 20.7, 20.8, 25.3, 29.2, 29.4, 32.5, 32.7, 34.4, 36.3, 37.9, 39.3, 40.0, 40.6, 44.4, 47.2, 63.6, 69.4, 71.1, 87.2, 91.3, 116.0, 125.0, 132.7, and 139.4.
~$ (NaCl): v 3470 (O-H), 2940 (C-H), 2370 (C_C), and 1690 (C=C).
(DEI) : m/z 426 (M*, 38%) , 408 (42) , 391 (27) , 390 (77), 261 (28), 259 (21), 219 (22), 195 (20), 181 (22), 179 (20) , 167 (21) , 165 (26) , 131 (23) , 129 (24) , 128 (20) , 115 (25), 105 (26), 91 (26), 83 (32), 69 (30), 59 (base), 55 (45), 45 (47), and 43 (86).
Fxa _t Mass (DEI) : calculated for CZBH42O3: m/z 426.3134.
Found: m/z 426.3123.
Preparation of analog LO, (14R,15S)-14,15-methano-1a,25-Dihydroxyvitamin D3 (10) A stirred mixture of dienyne 8 (38.6 mg, 0.0905 mmol), Lindlar catalyst (112 mg), and quinoline (312 JCL, 0.17 M in hexanes) in methanol (5 mL) was exposed to a positive pressure of hydrogen gas for 30 min. The mixture was filtered and concentrated to afford a residual oil which was purified by flash chromatography (elution with 80% EtOAc/hexane) to, afford 38.6 mg of the crude previtamin 9. IH-NMR analysis of the latter material showed the complete absence of starting material. A solution of the crude 9 {38.6 mg, 0.0905 mmol) in acetone (4 mL) was placed in a screw-capped vial and heated for 4 h in a constant temperature bath set at 80°C. The residue was concentrated under vacuum and purified by HPLC
(80% EtOAc/hexane, 4 mL/min, Rainin Dynamax 60 A column) to afford after vacuum drying 21.6 mg (56%) of the vitamin 10 (Analog LO) and 9.7 mg (25%) of the previtamin form (9).
~ (300 MHZ, CDC13): b -0.08 (dd, J'7.6, 3.7 Hz, 1H, H8) , 0.70 (apparent t, J'3.2 Hz, 1H, He) , 0.74 (s, 3H, C18-Me) , 0.80-2.00 (remaining ring and side chain hydrogens, series of m) , 0.86 (d, J'6. 5 Hz, 3H, C21-Me) , 1.20 (s, 6H, C26,27-2Me) , 2.28 (dd, J'13.4, 6.9 Hz, 1H), 2.58 (dd, J'13.4, 3.5 Hz, 1H), 2.75 (dt, J'13.4, 2.9 Hz, 1H), 4.21 (m, 1H, H3), 4.40 {apparent t, J'5.8 Hz, 1H, Hl) , 4.93 {s, 1H, H19) , 5.30 (s, 1H, H19) , 5.90 (dd, J'11.4, 1.4 Hz, 1H, H6 or H7)w, and 6.29 (d, J'11.4 Hz, 1H, H6 or H~) .
gy (100% EtOH): 1~"ax 268 rim (e 23,300), 1~" 230 rim (e 14,100).
(FAB+, EtOH/NBA): m/z 451 (MNa+, 4%), 345 (NBA+K, 8), 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) .
fact Mass (FAB+, EtOH/NBA) : calculated for C28H4qO3Na (MNa+): m/z 451.3188. Found: m/z 451.3174.
RXAMpLE 24 ygand Receptor Competition Assav This example describes a ligand receptor competitive assay used for determination of an analog's relative ability to bind to VDR"u~ expressed as relative competitive index (RCZ) .
The relative affinity of nonradioactive 1a,25(OH)ZD3 and each analog to compete with ['H]1a,25(OH)ZD3 for binding to the VDRn"~ of NB4 cells was carried out in vjtro. The NB4 cells were collected from a fast growing stage and the cellular VDRnuc of la, 25 (OH) ZD3 were extracted from KTED buffer containing 10 mM Tris-HCI, pH 7.4, 300 mM KC1, 1mM EDTA-and mM DTT. After sonication, the cell extract was further centrifuged at 500 x g for 10 min. The supernatant was 5 collected for use in a ligand-receptor binding assay.
In this assay, increasing concentrations ( 10-1° to 10-6 M) of nonradioactive 1a,25(OH)ZD3 or the tested analogs were incubated with NB4 cell extracts in the presence of a fixed saturating amount of 1 pmole of ['H] la, 25 (OH) ZD3. The reciprocal of the percentage of maximal binding of [3H] la, 25 (OH) 2D3 was then calculated and plotted as a function of the relative analog concentration versus [3H]1a,25(OH)2D3.
Each analog showed a linear plot and the slope of each curve represents the analog's competitive index value. The competitive index value for each analog is then normalized to the competitive index value of the radioactive ['H]1a,25(OH)ZD3, thereby generating the value of Relative Competitive Index (RCI) where the RCI for 1a,25(OH)ZD, is deffined as 100%.
2 0 The full description of the assay is f ound in rip*'h~~ s In F, ~'~..«. yitamins and Co-EnzYme~, Vol 67 494-500 , Academic Press, NY(1980); B~ochem Bionhvs Res Comm__un., 91:
827-834 (1979); and Fndocrinoloav. 139(2): 457-465 (1998).
FXI~MpLE 2 5 v' i-.;,~ i= D-Bindina Dr~t°," As av Reiat~ve Combetit.~ve Index This example describes a Relative Competitive Index Assay used for determination of analogs binding affinity to vitamin D-binding protein.
Binding of the 1,25(OH)ZD3 and its analogs to the human vitamin D-binding protein (hDBP) was performed at 4°C
essentially as described previously in the .TOUrnal of Bio~o ~ca'1 Chemistry 267; 3044-3051 (1992). One pmole of [3HJ 25 (OH) ZD3 and increasing concentrations of 1a, 25 (OH) zD3 or its analogs {10 1° to 10 6M) were added in 5 ~ul of ethanol into glass tubes and incubated with hDBP (0.18 ~M) in a final volume of 1 ml (0.01 M Tris-HC1, 0.154 M NaCl, pH 7.4) for 4 h at 4°C. Phase separation was then obtained by the addition pCTItjS98/19862 of 0.5 ml of cold dextran-coated charcoal.
The data was plotted as [competitor] / [ [sH] 25 (OH) D3] - vs.
1/[fraction bound]. The RCI was calculated as [slope of competitor]/[slope for 25(OH)D3] x 100. Results are seen in Figure 7. Although each analog was assayed in competition with [3H]25(OH)D3, the data are expressed as relative to the binding of 1a,25(OH)ZD3, with its RCI set to 100. In this assay, when the RCI of 1a,25(OH)ZD3 is set as 100, the RCI for 25(OH)D3 = 66,700.
FXAMPT.E 2 6 Tn Vivo Assays of Integ~i~~ ~''~""" Aheor~l ion and Bone Calcium Mobilization This example describes assays used for determination of analogs biological activity in intestinal calcium absorption (ICA) and bone calcium mobilization (BCM) assays.
ICA and BCM were measured in vivo in the vitamin D-deficient chick model system according to y~ochern PharmacoL, 18: 2347 (1969).
Twelve hours before assay, the chickens, which had been placed on a zero-calcium diet 48 h before assay, were injected intramuscularly with the vitamin metabolite 1a,25(OH)2D3 or analog (1 - 10,000 pmoles) dissolved in 0.1 mL of ethanol/1,2 propanediol (1:1, v/v). At the time of assay, 4.0 mg of 4°Ca2+
+ 5 ~Ci of 45Ca2+ (New England Nuclear) were placed in the duodenum of the birds lightly anesthetized with ether. After min, the birds were decapitated and the blood was collected.
The radioactivity content, which is a measure of ICA, of 0.2 mL of serum was measured in a liquid scintillation counter 30 {Beckman LS8000) to determine the amount of 45Ca2+ absorbed.
BCM activity was estimated from the increase of total serum calcium concentration, as determined by atomic absorption spectrophotometry.

~P~1 Differentiation Assav This example describes the cell differentiation assay and general conditions used for culturing HL-60, MCF-7, COS-7 and MG-63 cells. The details of the assay are described in sL...

Biol. Chem., 268: 13811-13919 (1993).
HL-60 cells were seeded at 1.2 x 105 cells/ml,-and 1,25(OH)2D3 or its analogs were added in ethanol in final concentration < 0.2%, in RPMI 1640 medium supplemented with 5 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, and 100 units/ml of streptomycin (Boehringer).
After 4 days of culture in a humidified atmosphere of 5% C02 in air at 37°C, the dishes were shaken to loosen any adherent cells. All cells were then assayed for differentiation by NBT
1o reduction assay and for proliferation by [3H]thymidine incorporation. Results are seen in Figure 10.
The COS-7 cells in Dulbecco's medium supplemented with 10% fetal calf serum (FCS) were seeded into 6-well plates to reach 40-60% confluence. After 24 h the medium was removed 15 and refreshed with culture medium containing 2% dextran-coated charcoal-treated FCS. The cells were then cotransfected with the pSGShVDR expression plasmid (1.5 ~tg) and the 1a,25(OH)ZD3 responsive element (VDRE) linked to the reporter plasmid (CT4)9TKGH (1.5 fig). The cells were then exposed to different 20 concentrations (10-il to 10-fim) of la, 25 (OH) ZD3 or analogs. The medium was assayed for the expression of human growth hormone using a radioimmunoassay.
MCF-7 cells were cultured in Dulbecco's minimal essential medium (DMEM) nutrient mix F12 (HAM) medium supplemented with 25 10% heat inactivated FCS, glutamine (2 mM) , penicillin (i00 units/ml) and streptomycin (0.1 mg/ml). Cultures were maintained at 37°C in a humidified atmosphere of 5% COZ in air.
MCF-7 cells were seeded at 5000 cells/well in the above-described medium in a 96-well microtiter plate in a final 30 volume of 0.2 ml per well. Triplicate cultures were performed. After 24h, 1a,25(OH)ZD3 or analogs were added in the appropriate concentrations from about 10-11 to about 10-6M
for an incubation period of 72 h. Then 1 ~CCi of [3H]thymidine was added to each well and the cells were harvested after a 35 4 h incubation with a Packard harvester and measured by the Packard Topcount System (Packard, Meriden, NH).
The MG-63 cells were seeded at 5 x 103 cells/ml in 96-well flat-bottomed culture plates (Falcon, Becton Dickinson, NJ) in a volume of 200 ul of DMEM containing 2% of heat-inactivated charcoal-treated fetal calf serum and 1,25(OH)ZD3 or its analogs were added in ethanol in final concentration < 0.2%. After 72 hrs of culture in a humidified atmosphere 5 of 5% COZ in air at 37°C, the inhibition of proliferation by ['H]thymidine incorporation and measurement in the medium of osteocalcin concentration using a homologous human RIA.
Nitro blue tetrazolium (NBT) reduction assay was according to J. Biol. Ch~;~, 267: 3044-3051 (I992). Superoxide ZO production was assayed by vitro blue tetrazolium-reducing activity as follows.
HL-60 cells at 1.0 x 105 cells/ml were mixed with an equal volume of freshly prepared solution of phorbol 12-myristate 13-acetate (200 ng/ml) and vitro blue tetrazolium 15 (2 mg/ml) and incubated for 30 min at 37°C. The percentage of cells containing black formazan deposits was determined using a hemacytometer.
E~~PLE 28 Transcaltachia Assav 20 This example describes the assay used for testing rapid response transcaltachia described in J. Biol. Chem. 268:
13811-13819 (1993).
White Leghorn cockerels (Hyline International, Lakeview, CA) were obtained on the day of hatch and maintained on a 25 vitamin D-supplemented diet (1.0% calcium and 1.0% phosphorus;
O. H. Kruse Grain and Milling, Ontario, CA) for 5-6 weeks to prepare normal vitamin D3-replete chicks for use in the transcaltachia studies.
Measurements of 45Ca2+ transport were carried out in 30 perfused chick duodena. Normal vitamin D-replete chicks weighing approximately 500 g were anesthetized with 0.3 ml per 100 g Chloropent (Fort Dodge, IA), and the duodenal loop was surgically exposed. The celiac vein and blood vessels branching off from the celiac artery were ligated before 35 cannulation of the celiac artery itself, and vascular perfusion was immediately initiated. Both the celiac artery and vein of the duodena were perfused with modified Grey's balanced salt solution (GBSS) + 0.9 mM Ca2+ which was oxygenated with 95% OZ and 5% CO2. A basal transport rate was established by perfusion with control medium for 20 minutes after the intestinal lumen was filled with °SCa2+. The tissue was then exposed to la, 25 (OH) ZD3 or analogs or reexposed to 5 control medium for, an additional 40 minutes. The vascular perfusate was collected at 2 min intervals during the last 10 min of the basal and during the entire treatment period.
Duplicate 100 ~1 aliquots were taken for determination of the °SCa2+ levels by liquid scintillation spectrometry. The results 10 are expressed as the ratio of the °SCa2+ appearing in the 40 min test period over the average initial basal transport period as seen in Figure 11.

MAP-kinase Activi~v 15 This example describes assays used for measurement of MAP-kinase activity in NB4 cells.
The detailed descriptions of the procedures are found in Journal of Ce11L1_a__r BloC1'~pmictrv~ in press, and in Endocrinoloav,139:457-465 (1998).
20 dell culture of NB4 cell N84 cells were obtained from Dr. K. A. Meckling-Gill {Guelph, Ont., Canada), and were originally isolated from a human patient with acute promyelocytic leukemia (APL) by Dr.
Michel Lanotte at the Hospital Saint-Louis (Unite INSERM 301, 25 Paris, France). The cell line is characterized by a translocation involving chromosomes 15 and I7, which is typical of the classical form of APL-M3 in the French-American-British [FAB] classification. NB4 cells were cultured in DMEM/F12 medium with 10% FCS at 5% C02 balanced 30 air and were routinely passaged as suspension cultures and only passages 8 to 20 were used for each assay. Cell growth and viability were assessed using the trypan blue dye exclusion method and 95% of the cells showed viability in the experiment culture conditions.
35 'rnLmLnor~reci ni tats on of Tvroa i nP-phosyhorvlated Proteins NB4 cells were cultured in 60-mm diameter dishes and treated with 1a,25(OH)2D3 or analogs in 4 ml of DMEM/F12 containing 10% charcoal-stripped FCS. At the end of the incubation period, cells were washed once in cold PBS
containing sodium vanadate at the concentration of 100 ~cM-and further extracted with RIPA buffer containing 50 mM Tris-HC1, pH 7.4; 150 mM NaCl, 0.2 mM Na3V04, 2 mM EGTA, 25 mM NaF, 1 Mm 5 PMSF, 0.25% sodium deoxycholate, 1% NP40, 2 ~ug/ml leupeptin, 2 ~cg/ml aprotinin and 2 ug/ml pepstatin.
Insoluble material was removed in a microcentrifuge at 14,000 rpm for 10 min. Protein concentration was determined with a protein assay kit (Bio-Rad Lab, Hercules, CA). For 10 immunoprecipitation, the supernatant was incubated with bead conjugated monoclonal anti-phosphotyrosine antibody overnight at 4°C. The immunoprecipitates containing the tyrosine phosphorylated proteins were washed four times with freshly prepared RIPA buffer and further eluted with 2X Laemmli gel 15 buffer.
At this point, the samples were either stored at -20°C
for further use or processed via Western blots. Equal loading of MAP-kinase protein was determined by running the Western blots using polyclonal anti-p42"~pk antibody. For this 20 purpase, samples were aliquoted from each cell extract before immunoprecipitation.
SDS Gel ElectrQ~ rpc;is arm Western blot Anti-phosphotyrosine immunoprecipitates of cell extract were resolved on 7.5% SDS-PAGE and transferred to PVDF
25 membranes according to the manufacturer's instructions (Amersham, Arlington Heights, IL). The membrane was further immunoblotted using a rabbit anti-p42'°°p'' polyclonal antibody overnight at 4°C followed by incubation with secondary horseradish peroxidase-conjugated mouse anti-rabbit antibody 3o for 1 hr at 25°C. The phosphorylated MAP-kinase bands were then visualized by enhanced chemiluminescence (ECL). A
Ultrascan LX Laser Densitometer (LKB, Bromma, Sweden) scanned the density of the immuno-phosphoprotein bands. The results were normalized by protein loading and further plotted as 35 percent of control of the band density. The specificity of p42'~ap'' phosphorylation was determined by resolving the tyrosine-phosphorylated proteins in SDS-PAGE, transferring the proteins to PVDF membrane and then incubating the membrane with anti-p42'""pk polyclonal antibody that had or had not been pre-exposed to MAP-kinase peptide for two hours.
MAP-kinase Activity in Chick Intes in~1 Cells Enterocytes were exposed either to 1a,25(OH)ZD3 (0.01-10 nM) for 1 min, 1,25(OH)ZD3 (1 nM) for 30 sec-5 min, or vehicle ethanol at 37°C. In some experiments, cells were pretreated with genistein (100 ~tM x 10 min). Lysates were prepared and MAP-kinase (p42 and p44) was immunoprecipitated from cell lysates as described above.
10 After three washes in immunoprecipitation buffer and two washes in kinase buffer {10 mM Tris-HC1, pH 7.2, 5 mM MgCl2, 1 mM MnCl2, 1 mM dithiothreitol, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulphonyl fluoride, 20 ~cg/ml leupeptin, 20 ug/ml aprotinin and 20 ~.g/ml pepstatin), immune complexes were 15 incubated at 37°C for 10 min in kinase buffer (50 ~C1/sample) containing myelin basic protein as an exogenous substrate for MAP-kinase (20 ug/assay) , 25 ACM ATP, and [Y'ZP]-ATP (2.5 ~cCi/assay). To terminate the reaction, the phosphorylated protein product was separated from free [y'2P]-ATP on ion-20 exchange phosphocellulose filters (Whatman P-81). Filters were immersed immediately in ice-cold 75 mM H3PO" washed (1 x 5 min, 3 x 20 min) and counted in a scintillation counter.
ExB~' pI,E 3 0 Treatment of O~tpnpornsic 25 This example shows method of treatment of osteoporosis using analogs of the invention, regimen and diagnostic evaluation of the disease progress.
Elderly patient suffering from pain in the bones is diagnosed with uncomplicated primary osteoporosis. Serum 30 calcium, phosphorus, alkaline phosphatase levels, protein electrophoresis patterns are nonaal. The patient has, however, a low urinary calcium excretion rate of less than 75 mg/day which does not increase with calcium supplementation. On X-ray examination, the vertebrae show decreased radiodensity due 35 to loss of trabecular structure.
The patient is diagnosed with osteoporosis and with impairment of calcium absorption. The patient is treated with 1-2 g of supplementary calcium and With 1-l0 micrograms/day of orally formulated 14,15-methano-1a,25(OH)ZD3, analog LO.
EKA_MPLE ~ 1 _ Treatment of Vita~n;n n-Dependent Rickets Type I
This example shows method of treatment of rickets using 5 the analog of the invention, regimen and diagnostic evaluation of the disease progress.
A child patient has visible abnormalities associated with rickets. Legs bowing is apparent in the femora and tibiae.
The ends of these bones are flaring at the knees.
10 The child is diagnosed with rickets after a deficiency in renal production of 1,25(OH)2D is discovered.
The child is put on a daily regimen of 1-to micrograms of analog EV formulated as drops until the swelling decreases and the bone mineralization is brought under control.
15 E~~AMPLE 3 2 Treatment o Ps~r;a~;~a This example shows the method of treatment of psoriasis using analogs of the invention and diagnostic evaluation of the disease process.
2o A patient is diagnosed with psoriasis on the basis of visual observation by a dermatologist of the presence of an external epidermis of silvery scaly papules and plaques.
The patient is provided with a topical cream containing 10 - 1000 ~g/gram of the analog of the invention. The cream 25 is used at the sites) of the psoriasis. The topical treatment is administered and continues until the psoriatic condition is alleviated.

Claims (23)

WHAT IS CLAIMED IS:

1. A compound of the formula I
Wherein R1 is hydrogen or hydroxy and wherein R1 on C1 and hydroxyl on C3 are positional isomers .alpha. and .beta. which may be the same or different in .alpha.-.alpha., .beta.-.beta., .alpha.-.beta. or .beta.-.alpha.
configuration;
wherein C5-C6 double bond is cis or trans;
wherein C7-C8 double bond is cis or trans;
wherein C14 hydrogen is .alpha. or .beta.;
wherein C16-C17 is a single or double bond;
wherein R2 is CH3 or CH2OH;
wherein R3 is a substituent selected from the group consisting of substituents with the proviso that when R1 is CH3 and when C1 and C3 are .alpha.-.beta., then R2 is not the substituent I-1, I-2, I-3, I-9 or I-10; or when C1 is in the .alpha. orientation and C3 is in the .beta.
orientation, C5-C6 double bond is cis or trans and C7-C8 double bond is trans, R1 is CH3, C14 hydrogen is in the .alpha.
orientation, C16-C17 is a single or double bond, then R2 is not the substituent I-1, I-2, I-3, I-4, I-5, I-9 or I-10; or when C1 is in the .beta. orientation, C3 is in the .beta.
orientation, C5-C6 double bond is cis, C7-C8 double bond is trans, R1 is CH3, C14 hydrogen is in a orientation, C16-C17 is a single bond, then R2 is not the substituent I-1; or when C1 is in the .alpha. orientation, C3 is in the .beta.
orientation, C5-C6 double bond is cis, C7-C8 double bond is trans, R1 is CH2OH, C14 hydrogen is in the .alpha.-orientation, C16-C17 is a single bond, then R2 is not the substituent I-1;
when C3 is in the .beta. orientation, C1 is not hydroxyl, C5-C6 double bond is cis, C7-C8 double bond is trans, R1 is methyl, C14 hydrogen is in the a orientation, C16-C17 is a single bond, then R2 is a substituent I-7 or I-8; and when C3 is in the .beta. orientation, C1 is in the .alpha.

orientation, C5-C6 double bond is cis, C7-C8 double bond is traps, R1 is CH3, C14 hydrogen is in the .alpha.-orientation, C16-C17 is a single bond, then R2 is a modified version of side chain I-6 wherein the C22 methylene (CH2) is replaced by a carbon-carbon triple bond.

2. A compound of the formula II
wherein C1 and C3 are positional isomers .alpha. and .beta. which may be the same or different in .alpha.-.alpha., .beta.-.beta., .alpha.-.beta.
or .beta.-.alpha.
configuration;
wherein C9 hydrogen and C10 methyl are positional isomers .alpha. and .beta. which may be the same or different in .alpha.-.alpha., .beta.-.beta., .alpha.-.beta.
or .beta.-.alpha. configuration;
wherein C16-C17 is a single or double bond;
wherein R1 is a substituent selected from the group consisting of substituents II-1 through II-10 or a pharmaceutically acceptable salt thereof;

with the proviso that when C1 and C3 are .alpha.-.beta., C9 and C10 are .alpha.-.alpha., .beta.-.beta., .alpha.-.beta. and .beta.-.alpha., and C16-C17 is a single bond, then R1 is not the substituent II-1.

3. A compound of the formula III
wherein C1 and C3 are positional isomers .alpha. and .beta. which may be the same or different in .alpha.-.alpha., .beta.-.beta., .alpha.-.beta.
or .beta.-.alpha.
configuration;
wherein C14 hydrogen is .alpha. or .beta.;
wherein C16-C17 is a single or double bond;
wherein R1 is a substituent selected from the group consisting of substituents when C1 and C3 hydroxyls are .alpha.-.beta. and C14 hydrogen is .alpha.
and C16-C17 is a single or double bond, then R1 is not the substituent III-4 and III-5, or a pharmaceutically acceptable salt thereof, with the proviso that when C1 and C3 hydroxyls are in .alpha.-.beta.
configuration, C14 hydrogen is a and C16-C17 is single bond, then R1 is not the substituent III-1, III-2, III-3, III-9, III-10; or -when C1 and C3 hydroxyls are .alpha.-.beta. and C14 hydrogen is a and C16-C17 is a single or double bond, then R1 is not the substituent III-4 and III-5.

4. A compound of the formula IV
wherein C1 and C3 are positional isomers .alpha. and .beta. which may be the same or different in .alpha.-.alpha., .beta.-.beta., .alpha.-.beta.
or .beta.-.alpha.
configuration;
wherein the C5-C6 is in .alpha. or .beta. configuration;
wherein C14 hydrogen is .alpha.;
wherein C16-C17 is a single or double bond;
wherein R1 is a substituent selected from the group consisting of substituents or a pharmaceutically acceptable salt thereof.

5. A compound of having a general formula V
wherein C1 and C3 are positional isomers .alpha. and .beta. which may be the same or different in .alpha.-.alpha., .beta.-.beta., .alpha.-.beta.
or .beta.-.alpha.
configuration;
wherein C5-C6 double bond is cis and C7-C8 double bond is traps; and wherein R1 is a substituent selected from the group consisting of substituents or a pharmaceutically acceptable salt thereof.

6. A method for treatment of diseases connected with or caused by vitamin D3 deficiency or overproduction, by providing a subject in need of such treatment a vitamin D3 analog which is either an agonist of a vitamin D3 receptor 148~

VDR nuc or VDR m~m, or its antagonist, wherein the analog is selected from the group consisting of compounds listed in Table 1.

7. The method of claim 6 wherein the disease is rickets, osteomalacia, osteoporosis, osteopenia, osteosclerosis or renal osteodystrophy, psoriasis, medullary carcinoma, Alzheimer's disease, hyperparathyroidism, hypoparathyroidism, pseudoparathyroidism, secondary parathyroidism, diabetes, cirrhosis, obstructive jaundice or drug-induced metabolism, glucocorticoid antagonism, hypercalcemia, malabsorption syndrome, steatorrhea, chronical renal disease, hypophosphatemic vitamin D-resistant rickets, vitamin D-dependent rickets, rickets type I, rickets type II
sarcoidosis, leukemia, prostate cancer, breast cancer, colon cancer, organ transplantation or an immunodisorder.

8. The method of claim 7 wherein the disease is osteoporosis, osteomalacia, rickets, renal osteodystrophy, hyperparathyroidism, hypercalcemia, rickets type I and rickets type II.

9. The method of claim 8 wherein the analog is a conformationally flexible agonist.

10. The method of claim 9 wherein the analog is selected from the group consisting of analogs listed in Table 2.

11. The method of claim 9 wherein the analog is selected from the group consisting of analogs listed in Table 3.

12. The method of claim 8 wherein the analog is conformationally restricted agonist.

13. The method of claim 12 wherein the analog is selected from the group consisting of analogs listed in Table 4.

14. The method of claim 8 wherein the analog is an antagonist.

15. The method of claim 14 wherein the analog is a conformationally flexible antagonist.

16. The method of claim 15 wherein the analog is an analog listed in Table 5.

17. The method of claim 7, wherein the analog is administered in a dose equivalent to 0.5-25 ug of 1.alpha.,25(OH)2D3 per 70 kg of body weight in an oral dose.

18. The method of claim 17 wherein the disease is osteoporosis.

19. The method of claim 18 wherein the analog is conformationally flexible analog 14.alpha., 15.alpha.-methano-1.alpha.,25(OH)2D3 (LO), 22-(m-dimethylhydroxymethyl) phenyl-23, 24, 25, 26, 27-pentanor-la(OH)D3 (EV), 1.alpha.,18,25(OH)2D3 (HS) or 6-s-cis locked analog 1.alpha.,25(OH)2-lumisterol(JN).

20. The method of claim 19 wherein the disease is osteomalacia and rickets.

21. The method of claim 20 wherein the analog is conformationally flexible analog 14.alpha., 15.alpha.-methano-1.alpha.,25(OH)2D3 (LO), 22-(m(dimethylhydroxymethyl) phenyl-23, 24, 25, 26, 27-pentanor-1.alpha.(OH)D3 (EV), 1.alpha.,18,25(OH)2D3 (HS) or 6-s-cis locked analog 1.alpha.,25(OH)2-lumisterol (JN) administered in 0,625 µg or 0.5-1 µg for treatment of rickets or equivalent to 0.25-2 µg 1.alpha.,25(OH)2D3 per 70 km weight for treatment of osteomalacia.

22. A pharmaceutical composition comprising at least one analog of 1.alpha.,25-dihydroxyvitamin D3 selected from the group of analogs listed in Table 1 in admixture with an adjuvant, said analog present in an amount sufficient to treat vitamin D
disease.

23. The composition of claim 22 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, or sarcoidosis, leukemia, prostate cancer, breast cancer, colon cancer, organ transplantation or an immunodisorder.