New 1alpha,25-dihydroxy-19-norvitamin D3 compounds of high biological activity: synthesis and biological evaluation of 2-hydroxymethyl, 2-methyl, and 2-methylene analogues.

Article abstract of DOI:10.1021/jm9802618

New highly active isomers of the natural hormone 1alpha, 25-dihydroxyvitamin D3 possessing an exomethylene group at the 2-position were prepared in a convergent manner, starting with (-)-quinic acid and the corresponding (20R)- and (20S)-25-hydroxy Grundm

Full text of DOI:10.1021/jm9802618

4662
J . Med. Chem. 1998, 41, 4662-4674
New 1r,25-Dih yd r oxy-19-n or vita m in D3 Com p ou n d s of High Biologica l Activity:
Syn th esis a n d Biologica l Eva lu a tion of 2-Hyd r oxym eth yl, 2-Meth yl, a n d
2-Meth ylen e An a logu es
Rafal R. Sicinski,† J ean M. Prahl, Connie M. Smith, and Hector F. DeLuca*
Department of Biochemistry, College of Agricultural and Life Sciences, 420 Henry Mall, University of Wisconsin-Madison,
Madison, Wisconsin 53706
Received April 30, 1998
New highly active isomers of the natural hormone 1R,25-dihydroxyvitamin D3 possessing an
exomethylene group at the 2-position were prepared in a convergent manner, starting with
(-)-quinic acid and the corresponding (20R)- and (20S)-25-hydroxy Grundmann ketones. These
2-methylene-19-norvitamins were efficiently converted to the 2-methyl and 2-hydroxymethyl
derivatives, some of which exhibited pronounced in vivo biological activity. Configurations of
the A-ring substituents were determined by 1H NOE difference spectroscopy as well as by spin
decoupling experiments. It was established that the bulky methyl and hydroxymethyl
substituents at C-2, due to their large conformational free energies, occupy mainly equatorial
positions. Additionally, hydroxylation of the C(10)-C(19) double bond in 1R,25-(OH)2D3 was
performed, resulting in 1R,19,25-trihydroxy-10,19-dihydrovitamin D3 derivatives in which the
hydroxymethyl substituent at C-10, for steric reasons, is forced to occupy an axial position. In
consequence, the vitamin D3 analogues were synthesized in which the 1R-hydroxy group,
required for biological activity, is almost exclusively axially or equatorially oriented because
of stabilization of the single A-ring chair conformations. The relative ability of the synthesized
analogues to bind the porcine intestinal vitamin D receptor was assessed and compared with
that of the natural hormone. It was established that vitamins possessing the axial orientation
of the 1R-hydroxy substituent exhibit a significantly increased receptor binding affinity.
Compounds with a 2-methylene substituent showed selective calcemic activity profiles, being
extremely effective on bone calcium mobilization. 2R-Methyl-substituted vitamins proved to
be much more active in vivo than the corresponding epimers with 2â-configuration. All of the
2-substituted vitamins exhibited pronounced HL-60 differentiating activity, those 2R-substituted
in the 20S-series being especially potent. The present studies imply that the axial orientation
of the 1R-hydroxy group is necessary for biological activity of vitamin D compounds.
In tr od u ction
exhibiting strong binding affinities to vitamin D-binding
protein (DBP), we have recently prepared 2R- and 2â-
hydroxy analogues of 2.9 The synthesized vitamins 3
and 4, as well as their 2-alkoxy derivatives, showed
selective activity in stimulating intestinal calcium
transport while having little or no activity in bone
calcium mobilization. Interestingly, they also exhibited
a high HL-60 differentiating activity. Furthermore,
some other 2-substituted (with hydroxyalkyl and fluo-
roalkyl groups) calcitriol analogues have recently been
synthesized and reported to exhibit strong affinities to
DBP.10 All these studies indicate that binding sites in
vitamin D receptors and other binding proteins can
accommodate different substituents at C-2, present in
the synthesized vitamin D analogues.
The discovery of the hormonally active form of vita-
min D3, 1R,25-dihydroxyvitamin D3 (1R,25-(OH)2D3,
calcitriol, 1; Figure 1), has stimulated great interest in
establishing its physiological role.1,2 It is now well-
known that 1 not only regulates calcium homeostasis
and phosphorus metabolism in animals and humans,3
but it also affects the human immune system, inhibits
cell proliferation, and promotes cellular differentiation.4
The hormone has been successfully used in the treat-
ment of calcium and bone disorders5 and the skin
disorder psoriasis.6 Since large doses of 1 can cause
hypercalcemia, there is a considerable interest in aca-
demia and the pharmaceutical industry to prepare
noncalcemic calcitriol analogues that still retain anti-
proliferative activity. Such a desired selective activity
toward differentiation was found in the 19-nor analogue
2, synthesized in our laboratory.7 Prompted by our
result and literature data concerning interesting bio-
logical potency of 2-substituted calcitriol derivatives,8
In a continuing investigation of the structure-activity
relationship of the vitamin D molecule, we decided to
prepare 19-norvitamin D compound analogues with
2-methyl and 2-hydroxymethyl substituents in an effort
to alter the orientation of the 1R-hydroxyl functions. As
a convenient precursor of such analogues, we prepared
the 2-methylene compound, which by itself seemed to
be an interesting synthetic target. It is commonly
accepted that the presence of a 1R-hydroxy group in the
vitamin D molecule is the dominant factor in receptor
binding11 and it is required for the expression of
* To whom correspondence should be addressed: Dr. Hector F.
DeLuca. Telephone: 608-262-1620. Fax: 608-262-7122. E-mail:
deluca@biochem.wisc.edu.
† Present address: Department of Chemistry, University of Warsaw,
ul. Pasteura 1, 02-093 Warsaw, Poland.
10.1021/jm9802618 CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/22/1998

New 1R,25-(OH)2D3 with High Biological Activity
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4663
F igu r e 1. Chemical structure of 1R,25-dihydroxyvitamin D3 (calcitriol) and its analogues.
biological activity. An analogue of the natural hormone
1, 1R,25-dihydroxy-(5E)-vitamin D3 (5), in which the
exocyclic methylene group is transposed, in comparison
with 1, from the right to the left side of ring A exhibits
only ca. 8-fold lower binding to the receptor protein than
the hormone 1.11d Both isomers 1 and 5, in a purely
formal sense, can be considered as A-ring isomers
having the exocyclic methylene unit in the two alterna-
tive “ortho” positions in relation to the C(5)-C(6) double
bond. However, the third isomer is also possible, and
it was therefore of interest to synthesize compound 6a ,
characterized by the transposition of the methylene unit
to the “para” position, i.e., at C-2. The relatively small
2-methylene substituent should not interfere with bind-
ing to the vitamin D receptor, but it changes the
character of both (1R and 3â) cyclohexanediol A-ring
hydroxyls. Both are now in the allylic positions, simi-
larly as the 1R-hydroxy group, crucial for biological
activity, in the natural hormone 1.
This paper describes our synthetic route to the
isomeric (at C-20) 1R,25-dihydroxy-2-methylene-19-nor-
vitamin D3 compounds 6a ,b and their conversion to the
respective 2-methyl and 2-hydroxymethyl derivatives
7a ,b-10a ,b. The calcemic and cellular differentiation
activities of these new 19-norvitamins were tested, and
a strong influence on biological potency of their A-ring
conformations was observed, strongly suggesting that
the 1R-hydroxyl must be in the axial orientation for
maximal biological activity. Further, the (20S)-2R-
methyl-substituted compound 7b and 2-methylene de-
rivatives 6a ,b show strong preferential activity on bone.
Additionally, two vitamin D analogues (13 and 14)
with hydroxymethyl substituents at C-10 were pre-
pared, being 19-hydroxy derivatives of 1R,25-dihydroxy-
10,19-dihydrovitamin D3 compounds 11 and 12, syn-
thesized by us previously.12
Resu lts a n d Discu ssion
Ch em istr y. The strategy of our synthesis of 2-sub-
stituted 19-norvitamins was based on the Wittig-
Horner coupling, pioneered by the Lythgoe group13 and
successfully used by us in the recent preparation of 3
and 4. Since the corresponding C,D-ring ketones were
available,9,14 we focused our attention on the synthesis
of the phosphine oxide A-ring synthon.
The secondary hydroxy group of the methyl quinicate
derivative 15 (Scheme 1), easily prepared from com-
mercial (1R,3R,4S,5R)-(-)-quinic acid,7a was oxidized
with ruthenium tetroxide in a catalytic process employ-
ing RuCl3 and NaIO4. The use of such a strong oxidant
was necessary for an effective oxidation of this very
hindered alcohol. Other, more commonly used reagents
can also be employed (e.g., pyridinium dichromate), but
the reaction usually requires much longer time for
completion. The next step of the synthesis comprises
the Wittig reaction of the sterically hindered 4-keto
compound 16 with an ylide prepared from methyltriph-
enylphosphonium bromide and n-butyllithium. Our
numerous attempts did not succeed in improving the
yield (24%) of the desired 4-methylene product 17. Also,
attempted reaction of 16 with the PO-ylide obtained
from methyldiphenylphosphine oxide upon deprotona-

4664 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Sicinski et al.
Sch em e 1
tion with n-butyllithium, the reagent recommended for
methylenation of sterically hindered ketones,15 resulted
in formation of a complex mixture of products. Rather
unexpectedly, the following reduction of a carbomethoxy
group in 17 with diisobutylaluminum hydride (DIBALH)
proceeded as poorly as in the case of the previous
synthetic step; however, the use of lithium aluminum
hydride as a reducing agent doubled the yield of 18.
Without any further problems, this vicinal diol was
cleaved by sodium periodate, and the resulting cyclo-
hexanone derivative 19 was smoothly converted to the
unsaturated ester 20 by the Peterson olefination with
methyl (trimethylsilyl)acetate. On the basis of litera-
ture precedent13c and our experience,7a,9 compound 20
was reduced with DIBALH to the allylic alcohol 21
which was in turn tosylated in situ with n-butyllithium
and p-toluenesulfonyl chloride, converted into the cor-
responding phosphine by a reaction with diphenylphos-
phine lithium salt, and oxidized with hydrogen peroxide
to the final A-ring phosphine oxide 22.
The Wittig-Horner coupling of the conjugate base of
22, generated upon deprotonation with n-butyllithium,
with the protected 25-hydroxy Grundmann’s ketone
23a , prepared according to the published procedure,9
produced the expected protected 19-norvitamin D com-
pound 24a in a high yield. This, after quantitative
deprotection with AG 50W-X4 cation-exchange resin,
afforded 1R,25-dihydroxy-2-methylene-19-norvitamin D3
(6a ).
In our previous paper we demonstrated that 10,19-
dihydro derivatives 11 and 12 can be efficiently obtained
by selective reduction of 1; similar selectivity was
observed in the case of the isomer 5 with a (5E,7E)-5,7,-
10(19)-triene system.12 It turned out that the selective

New 1R,25-(OH)2D3 with High Biological Activity
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4665
Sch em e 2
hydrogenation of the A-ring exomethylene unit is pos-
sible also with compound 6a , where it is located in the
“para” position in relation to the C(5)-C(6) double bond.
Thus, homogeneous catalytic hydrogenation of 6a per-
formed in the presence of tris(triphenylphosphine)-
rhodium(I) chloride [Wilkinson’s catalyst, (Ph3P)3RhCl]
provided efficiently an equimolar mixture of 2-methyl-
19-norvitamins 7a and 8a (Scheme 2), easily separated
by HPLC. A similar chemoselectivity was also observed
in hydroboration reactions. We employed for this
purpose 9-borabicyclo[3.3.1]nonane (9-BBN) as a re-
agent and reaction conditions analogous as those used
by Okamura for hydroboration of simple vitamin D
compounds.16 Since this literature precedent concerned
hydroboration of 1-desoxy compounds, namely, 5E- and
5Z-isomers of vitamins D2 and D3, we first tested the
process using 1 as a model compound. The formed
organoborane intermediate (1/9-BBN adduct) was sub-
sequently oxidized with basic hydrogen peroxide. The
best yield of the corresponding 10R- and 10â-hydroxym-
ethyl products 13 and 14, separated by flash chroma-
tography, achieved by us amounted to 12% and 9%,
respectively. Such hydroboration-oxidation conditions
allowed us also to hydroxylate exclusively the C(2)dCH2
unit in the vitamin 6a , leaving the intercyclic C(5)d
C(6)-C(7)dC(8) diene moiety unaffected. The isolated
epimeric mixture of 2-hydroxymethyl derivatives 9a and
10a (ca. 1:2, 35% yield) was purified and separated by
straight- and reversed-phase HPLC.
ketone,14 with the lithiated phosphine oxide 22 provided
19-norvitamin 24b, which after hydrolysis of the hy-
droxy-protecting groups gave (20S)-1R,25-dihydroxy-2-
methylene-19-norvitamin D3 (6b). This compound, like
its 20R-counterpart 6a , was subjected to homogeneous
hydrogenation with Wilkinson’s catalyst leading to
2-methyl analogues (7b, 8b) and underwent hydrobo-
ration with 9-BBN yielding 2-hydroxymethyl derivatives
(9b, 10b). In accord with the literature data concerning
similar systems,18 all of the synthesized vitamin D
analogues with the 20S-configuration (6b-10b) show
1
in their H NMR spectra signals of the 20-methyl group
which are shifted by ca. 0.083 ppm upfield with respect
to those of the corresponding 20R-isomers of “natural”
series. Stereochemistry at C-2 and C-10 in the synthe-
sized vitamin D compounds was tentatively assigned on
the basis of a simple conformational analysis, molecular
modeling studies, and especially 500-MHz 1H NMR
spectroscopy.
It has been commonly accepted19 that vitamin D
compounds in solutions exist as a mixture of two rapidly
equilibrating A-ring chair conformers abbreviated as R-
and â-forms (Figure 2a). Such conformational equilibria
have been found for vitamin D3, 25-hydroxyvitamin D3
(25-OH-D3), 1R-hydroxyvitamin D3 (1R-OH-D3), and the
natural hormone 1R,25-dihydroxyvitamin D3 (1R,25-
(OH)2D3, 1)20 as well as some other A-ring-substituted
vitamin D derivatives.16,21 Considering the conforma-
tional free energy (A value)22 of a methyl substituent
(1.7 kcal/mol)23 and a value of syn-clinal interaction
One of the interesting modifications in the vitamin
D side chain is the C-20 epimerization. It is well-known
that vitamin D analogues with “unnatural” stereochem-
istry of the methyl group at C-20 exhibit considerably
increased calcemic activity.17 Thus, condensation of the
protected C,D-ring synthon 23b, prepared by triethyl-
silylation of the parent (20S)-25-hydroxy Grundmann’s
between methyl and hydroxy group (0.35 kcal/mol),24
a
significant free energy advantage (1.35 kcal/mol) can be
easily computed for the corresponding conformers of the
synthesized 19-norvitamins 7 and 8, those possessing
an equatorial methyl group at C-2. It can be also
predicted that the A-ring conformational equilibrium for

4666 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Sicinski et al.
F igu r e 2. Conformational equilibrium in ring A of 1R-hydroxyvitamin D analogues (a) and 2-methylene-19-norvitamins 6 (b)
and the preferred, energy-minimized A-ring conformations of the synthesized analogues: 7 (c), 8 (d), 9 (e), 10 (f), 13 (g), and 14
(h). Hydrogen bonds are represented by dotted lines. The steric energy differences between the preferred conformers and their
partners with the inverted chair forms (calculated for model compounds lacking a side chain) are given. The corresponding
percentage populations (in parentheses) of conformers are given for room temperature (25 °C).
the vitamins 9 and 10, which have bulkier hydroxym-
ethyl substituents, should be even more biased toward
the conformers with the equatorial 2-CH2OH group.25
It has been established that in 10,19-dihydrovitamin
D compounds an additional strong interaction (4.84 kcal/
mol),12 usually termed as the A(1,3)-strain,26,27 takes
place between an equatorial 10-methyl and C(7)-H
group. Therefore, in the synthesized vitamins 13 and
14, a large energy gap of at least 3 kcal/mol can be
expected in favor of the A-ring chair conformer with an
axial 10-hydroxymethyl substituent, even if we allow
for some free energy decrease caused by the formation
of a hydrogen bond between the equatorial CH2OH
group and 1R-hydroxyl in the isomer 14 (where the
presence of two axial substituents at C-1 and C-10 in
the inverted chair form precludes hydrogen bond forma-

New 1R,25-(OH)2D3 with High Biological Activity
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4667
tion). Thus, it can be predicted that the large confor-
mational free energy of the 2-methyl or 2-hydroxyme-
thyl substituent in 19-norvitamins 7, 8 and 9, 10,
respectively, and the strong interaction between the 10-
hydroxymethyl and C(7)-H group in the 10(19)-dihydro
analogues 13 and 14 destabilize one A-ring chair
conformation and favor an alternate inverted chair form.
A careful analysis of 1H NMR spectra (in CDCl3) of the
synthesized vitamins (exemplified for 6a , 7a , and 13)
confirmed these predictions. The proportion of the two
rapidly equilibrated chair forms of ring A of 2-methyl-
ene-19-norvitamin 6a (Figure 2b) can be established by
the analysis of a multiplet pattern of the methylene
protons at C-4 and C-10, namely, from the magnitudes
of their degenerate couplings to the methine protons at
C-3 and C-1, respectively. The corresponding signals
of the equatorial protons at C-4 and C-10 were assigned
by 1H NOE difference spectroscopy, involving vinylic 6-
and 7-H, whereas the connectivity of the protons was
established by spin decoupling experiments. The ob-
served trans-vicinal coupling (6.0 Hz) of the 4â-proton
resonating at δ 2.33 and the analogous trans-coupling
(8.4 Hz) of the 10R-proton resonating at δ 2.29 represent
averaged axial-axial and equatorial-equatorial values.
From these data and the vicinal coupling constants
reported for cyclohexanol protons (J ax,ax ) 11.1 Hz, J eq,eq
) 2.7 Hz),28 the conformational equilibrium for 6a was
established to be ca. 6:4 in favor of the conformer that
has an equatorial 1R-OH. In the case of vitamin 7a , a
large half-width (24 Hz) of the 3R-H multiplet at δ 3.61
supports its axial disposition and indicates that two
axial-axial couplings must be involved, namely, those
with the protons at C-2 and C-4. Therefore, the 2R-
configuration of the equatorial methyl substituent in 7a
has been unequivocally assigned. Moreover, the mag-
nitudes of the vicinal coupling constants of the 4R- and
4â-H (4.3 and ca. 12 Hz, respectively)28 indicate that
compound 7a exists in the single A-ring conformation
limited to the compounds that can assume an A-ring
chair conformation in which the 1R-hydroxy group
occupies the equatorial orientation.30 Such conforma-
tion, according to this hypothesis, has the proper
geometry for binding to the protein receptor, a step
which is necessary to induce the biological response
leading to the calcium transport and calcium mobiliza-
tion in the body. However, our recent results of biologi-
cal testing of 1R,25-dihydroxy-10,19-dihydrovitamin D3
compounds did not support the idea that the most
equatorially favored 1R-hydroxyl would be the most
biologically active one. On the contrary, 1R,25-dihy-
droxy-10(S),19-dihydrovitamin D3 (12), the analogue
strongly biased toward the A-ring chair conformer
possessing the axially oriented 1R-hydroxy group, pro-
vided the greatest in vivo biological response and
showed very significant activity on intestinal calcium
transport.12 While this work was in progress, a com-
munication appeared on the synthesis and biological
activities of 2R- and 2â-methyl-1R,25-dihydroxyvitamin
D3 analogues.31 The J apanese authors conclude that
the introduction of a methyl substituent at C-2 shifts
either way, to a certain extent, the equilibrium between
the A-ring chair conformers, and the biological potency
of the synthesized vitamins is highly dependent on the
configuration of the 2-methyl group.
Recently, we have reported that 1R,25-dihydroxy-19-
norvitamin D3 derivatives, substituted at C-2 with
hydroxy (3 and 4) or alkoxy groups, showed selective
activity profiles by retaining their ability to cause
cellular differentiation and to increase intestinal cal-
cium transport while having markedly reduced the
ability to elevate plasma calcium at the expense of
bone.9 However, the complexity of their 1H NMR
spectra precluded any establishing of the A-ring con-
formational equilibria in these vitamins. Nevertheless,
the conformational analysis of some intermediates9 as
well as a low A value of the hydroxy substituent (0.52
kcal/mol)23 seems to indicate that the free energy
difference between the equilibrating A-ring chair forms
of the 2-hydroxy(alkoxy)-substituted 19-norvitamins
should not be remarkable.
1
with an axial 3R-H. In the H NMR spectrum of the
vitamin 13, the multiplet pattern of 1â-H, resonating
at δ 4.29 (∼dt, J ) 12.2, 4.3 Hz), clearly indicates its
axial orientation. The cis-relationship between this
proton and the equatorial proton at C-10, supported by
the vicinal coupling constant (4.3 Hz), establishes the
10R-orientation of the hydroxymethyl group.
In a continuing effort to explore the 19-nor class of
pharmacologically important vitamin D compounds, the
analogues which are characterized by the transposition
of the A-ring exocyclic methylene group, present in the
normal vitamin D skeleton at C-10, to carbon 2, i.e.,
2-methylene-19-norvitamin D compounds 6a ,b, have
been synthesized and tested. 2-Methyl (7a ,b and 8a ,b)
and 2-hydroxymethyl (9a ,b and 10a ,b) analogues of 19-
nor-1R,25-(OH)2D3 (2) were easily obtained from the
2-methylene precursors, and analogously 1R,19,25-tri-
hydroxy-10,19-dihydrovitamin D3 isomers (13, 14) were
prepared, all of them possessing the strongly preferred,
single chair conformation of ring A. It was, therefore,
possible to examine the effect of the orientation of the
1R-substituent on biological activities of the compounds
synthesized.
Molecular modeling was also employed to establish
the A-ring conformations of the synthesized compounds.
Force field calculations29 were carried out for model
compounds, analogues of vitamins 6-10, 13, and 14
lacking the steroidal side chain at C-17. The calculated
A-ring conformational populations (Figure 2c-h) were
in excellent agreement with our previous conformational
considerations and fully supported the strong bias
toward conformers with the equatorial configuration of
C(2)-substituents in vitamins 7-10 and preponderance
of the forms with an axial orientation of the hydroxym-
ethyl groups at C-10 in the corresponding derivatives
13 and 14. The results of our molecular modeling
studies indicate that in all but one (14) of the vitamins
possessing an A-ring hydroxymethyl group, the latter
substituent is involved in hydrogen bond formation with
the neighboring secondary hydroxyl.
The synthesized vitamins were tested for their ability
to bind the porcine intestinal vitamin D receptor. A
comparison between the natural hormone 1 and ana-
logue 6a indicates that the transposition of the exocyclic
methylene group from C-10 to C-2 does not result in a
change of the binding affinity (Table 1). Similarly,
Biologica l E va lu a t ion . In 1974, it was proposed
that the calcium regulation ability of vitamins D is

4668 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Sicinski et al.
Ta ble 1. VDR Binding Propertiesa and HL-60 Differentiating Activitiesb of 2-Substituted Analogues of
1R,25-Dihydroxy-19-norvitamin D3 and Their 20S-Isomers
VDR binding
HL-60 differentiation
compound
compd no.
ED50 (M)
binding ratio
ED50 (M)
activity ratio
1R,25-(OH)2D3
1
6a
6b
8.0 × 10-11
1.2 × 10-10
1.0 × 10-10
1
1.5
1.3
4.0 × 10-9
4.2 × 10-9
1.5 × 10-10
1
1
0.04
2-methylene-19-nor-1R,25-(OH)2D3
2-methylene-19-nor-(20S)-1R,25-(OH)2D3
1R,25-(OH)2D3
1
9.0 × 10-11
4.2 × 10-10
4.0 × 10-10
3.5 × 10-9
5.0 × 10-10
1
4.0 × 10-9
8.0 × 10-11
7.0 × 10-11
8.0 × 10-9
7.0 × 10-10
1
2R-methyl-19-nor-1R,25-(OH)2D3
2R-methyl-19-nor-(20S)-1R,25-(OH)2D3
2â-methyl-19-nor-1R,25-(OH)2D3
2â-methyl-19-nor-(20S)-1R,25-(OH)2D3
7a
7b
8a
8b
4.6
4.4
39
0.02
0.02
2.0
5.5
0.17
1R,25-(OH)2D3
1
9a
9b
10a
10b
6.0 × 10-11
8.0 × 10-10
8.0 × 10-11
7.0 × 10-9
5.0 × 10-10
1
13
1.3
117
8.3
4.0 × 10-9
2.0 × 10-8
2.0 × 10-9
1.0 × 10-7
4.5 × 10-9
1
2R-(hydroxymethyl)-19-nor-1R,25-(OH)2D3
2R-(hydroxymethyl)-19-nor-(20S)-1R,25-(OH)2D3
2â-(hydroxymethyl)-19-nor-1R,25-(OH)2D3
2â-(hydroxymethyl)-19-nor-(20S)-1R,25-(OH)2D3
5.0
0.5
25
1.1
a
Competitive binding of 1R,25-(OH)2D3 (1) and the synthesized vitamin D analogues to the porcine intestinal vitamin D receptor. The
experiments were carried out in triplicate on two different occasions. The ED50 values are derived from dose-response curves and represent
the analogue concentration required for 50% displacement of the radiolabeled 1R,25-(OH)2D3 from the receptor protein. Binding ratio is
b
the ratio of the analogue average ED50 to the ED50 for 1R,25-(OH)2D3. Induction of differentiation of HL-60 promyelocytes to monocytes
by 1R,25-(OH)2D3 (1) and the synthesized vitamin D analogues. Differentiation state was determined by measuring the percentage of
cells reducing nitro blue tetrazolium (NBT). The experiment was repeated three times. The ED50 values are derived from dose-response
curves and represent the analogue concentration capable of inducing 50% maturation. Differentiation activity radio is the ratio of the
analogue average ED50 to the ED50 for 1R,25-(OH)2D3.
2-methyl-substituted 19-norvitamins 7a ,b and 8b were
only 4-5 times less active than 1R,25-(OH)2D3, while
the 2â-methyl isomer in the 20R-series (8a ) was 39-fold
less effective. The 2R-(hydroxymethyl)vitamin D ana-
logue with the “unnatural” configuration at C-20 (9b)
was almost equivalent to the hormone 1 with respect
to receptor binding, and the isomeric 10b proved to be
less potent (6-8 times) than these compounds. The
corresponding 2R-hydroxymethyl analogue possessing
the “natural” 20R-configuration (9a ) was 10-fold less
effective than the 20S-compound 9b, whereas the 2â-
isomer 10a was 90 times less potent. The foregoing
results of the competitive binding analysis show that
vitamins with the axial orientation of the 1R-hydroxy
group exhibit a significantly enhanced affinity for the
receptor. Both isomeric 1R,19,25-trihydroxy-10,19-di-
hydrovitamin D3 compounds 13 and 14 were 1000 times
less potent than the natural hormone 1 in displacement
of the radiolabeled 1R,25-(OH)2D3 from the receptor
protein (data not shown).
Attention is drawn to the fact that both 2-methylene-
19-norvitamins 6a ,b, when tested in vivo in rats,
exhibited an extremely high ability to mobilize calcium
from bone, while having no intestinal calcium transport
activity (Table 2). It was not surprising that the 1R,-
19,25-trihydroxy-10,19-dihydrovitamin D3 compounds
13 and 14 were devoid of biological activity (data not
shown), in view of our previous results indicating low
potency of the parent 1R,25-dihydroxy-10,19-dihydro-
vitamin D3 isomers.12 However, vitamins possessing a
hydroxymethyl substituent at C-2 turned out to be
inactive, including those in the 20S-series (9b , 10b ).
Thus, on the basis of the results described above, it was
impossible to draw any conclusions regarding the con-
formation-activity relationship. On the contrary, the
in vivo biological testing of 2-methyl-substituted 19-nor-
1R,25-(OH)2D3 analogues appeared to be much more
interesting and informative. Table 2 shows that a 260-
pmol dose of 2R-methyl vitamin 7a is equal to or only
slightly less effective on intestinal calcium transport and
bone calcium mobilization than hormone 1. The overall
calcemic activity of its analogue 7b from the 20S-series
proved to be appreciably higher than that of 1R,25-
(OH)2D3. However, no elevation of serum calcium or
bone calcium mobilization was found for any of the 2â-
methyl analogues 8a ,b at this dose level. The foregoing
activity of the vitamin D analogues strongly depends
on the conformation of the A-ring. However, in any
case, these results do not support the suggestion that
the equatorially favored 1R-hydroxyl is required for
calcemic activity, because analogues with such an
orientation of the 1R-OH group show reduced biopotency
in both intestine and bone. Thus, the results indicate
a much greater biological activity of the vitamins
possessing the axial 1R-hydroxy substituent.
In the next assay, the cellular activity of the synthe-
sized compounds was established by studying their
ability to induce differentiation of human promyelocyte
HL-60 cells into monocytes. Interestingly, the transpo-
sition of the A-ring exocyclic methylene group to C-2
did not change the cellular differentiation ability of the
analogue 6a with respect to the parent hormone 1
(Table 1). It was found that, with an exception of an
equally active 10b, all of the synthesized vitamin D
analogues with the “unnatural” 20S-configuration were
more potent than 1R,25-(OH)2D3. Moreover, the same
relationship between cellular activity and conformation
of the vitamin D compounds was established as in the
case of receptor binding analysis and in vivo studies:
i.e., 2R-substituted vitamin D analogues were consider-
ably more active than their 2â-substituted counterparts
with the equatorially oriented 1R-hydroxy group. Thus,
2R-methyl vitamins 7a ,b proved to be 100 and 10 times,
respectively, more active than their corresponding 2â-
isomers 8a ,b in the cultures of HL-60 in vitro, whereas
in the case of 2-hydroxymethyl derivatives (9a ,b versus
10a ,b) these differences were smaller. Since vitamins
with a 2â-methyl substituent (8a ,b) and both 2-hy-
droxymethyl analogues in the 20S-series (9b, 10b) have
selective activity profiles combining high potency in
cellular differentiation and lack of calcemic activity,
such compounds are potentially useful as therapeutic

New 1R,25-(OH)2D3 with High Biological Activity
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4669
Ta ble 2. Support of Intestinal Calcium Transport and Bone Calcium Mobilization by 2-Substituted Analogues of
1R,25-Dihydroxy-19-norvitamin D3 in Vitamin D-Deficient Rats on a Low-Calcium Dieta
compd
no.
amount
(pmol)
Ca transport S/M
(mean ( SEM)
serum Ca
(mean ( SEM)
compound
none (control)
1R,25-(OH)2D3
2-methylene-19-nor-1R,25-(OH)2D3
0
260
130
260
130
260
5.5 ( 0.2b
5.1 ( 0.2b
1
6a
6.2 ( 0.4c
7.2 ( 0.5c
1
1
5.3 ( 0.4d
9.9 ( 0.2d
2
2
4.9 ( 0.6d
9.6 ( 0.3d
1
1
2-methylene-19-nor-(20S)-1R,25-(OH)2D3
6b
5.8 ( 0.8e
13.8 ( 0.5e
2
2
4.6 ( 0.7e
14.4 ( 0.6e
none (control)
1R,25-(OH)2D3
2R-methyl-19-nor-1R,25-(OH)2D3
0
260
130
260
130
260
2.3 ( 0.4b
3.9 ( 0.1b
1
7a
5.6 ( 0.6c
6.1 ( 0.2c
1
1
4.3 ( 0.6d
4.8 ( 0.1d
2
2
5.3 ( 0.6d
5.8 ( 0.3d
1
1
2â-methyl-19-nor-1R,25-(OH)2D3
8a
4.4 ( 0.2e
4.1 ( 0.1e
2
2
3.0 ( 0.4e
3.8 ( 0.1e
none (control)
1R,25-(OH)2D3
2R-methyl-19-nor-(20S)-1R,25-(OH)2D3
0
260
130
260
130
260
2.9 ( 0.3b
4.2 ( 0.1b
1
7b
4.6 ( 0.2c
6.6 ( 0.4c
1
1
12.9 ( 1.9d
8.3 ( 0.7d
2
2
8.4 ( 1.1d
10.3 ( 0.1d
1
1
2â-methyl-19-nor-(20S)-1R,25-(OH)2D3
8b
2.9 ( 0.3e
4.4 ( 0.1e
2
2
3.8 ( 0.1e
4.4 ( 0.1e
none (control)
1R,25-(OH)2D3
2R-(hydroxymethyl)-19-nor-(20S)-1R,25-(OH)2D3
0
260
130
260
130
260
4.0 ( 0.3b
3.8 ( 0.1b
1
9b
6.6 ( 0.5c
5.2 ( 0.1c
1
1
5.0 ( 0.3d
4.0 ( 0.1d
2
2
5.8 ( 0.4d
3.9 ( 0.1d
1
1
2â-(hydroxymethyl)-19-nor-(20S)-1R,25-(OH)2D3
10b
3.5 ( 0.7e
3.6 ( 0.1e
2
2
3.5 ( 0.3e
3.5 ( 0.2e
a
Weanling male rats were maintained on a 0.47% Ca diet for 1 week and then switched to a low-calcium diet containing 0.02% Ca for
an additional 3 weeks. During the last week, they were dosed daily with the appropriate vitamin D compound for 7 consecutive days. All
doses were administered intraperitoneally in 0.1 mL of propylene glycol/ethanol (95:5). Controls received the vehicle. Determinations
were made 24 h after the last dose. There were at least 6 rats/group. Statistical analysis was done by Student’s t-test. Statistical data:
serosal/mucosal (S/M) m, panel 1, b from c, p < 0.05, c from d1, d2, and e2, p < 0.025, b from d1, d2, e1, and e2, NS; panel 2, b from c, p <
0.001, b from d1, e1, and e2, NS, b from d2, p < 0.005; panel 3, b from c, p ) 0.05, b from d1 and d2, p < 0.001, b from e1 and e2, NS; panel
4, b from c, p < 0.001, b from d1, p < 0.01, b from d2, p < 0.001, b from e1 and e2, NS; serum calcium m, panel 1, b from c, p < 0.005, b
from d1, d2, e1, and e2, p < 0.001; panel 2, b from c, p < 0.005, b from d1, e1, and e2, NS, b from d2, p < 0.025; panel 3, b from c, p ) 0.05,
b from d1 and d2, p < 0.001, b from e1 and e2, NS; panel 4, b from c, p ) 0.001, b from all others, NS.
spectra were recorded at 500 MHz with a Bruker AM-500 FT
spectrometer in deuteriochloroform. Chemical shifts (δ) are
reported downfield from internal Me4Si (δ 0.00). Low- and
high-resolution mass spectra were recorded at 70 eV on a
Kratos DS-50 TC instrument equipped with a Kratos DS-55
data system. Samples were introduced into the ion source
maintained at 120-250 °C via a direct insertion probe. High-
performance liquid chromatography (HPLC) was performed
on a Waters Associates liquid chromatograph equipped with
agents for the treatment of cancer. As in the case of
the bioassays described previously, compounds 13 and
14 were found to be totally inactive also in the HL-60
system (data not shown).
Con clu sion s
These results as well as our previous studies on 19-
nor analogues of the hormone 1R,25-(OH)2D3 indicate
that variation of substituents on C-2 in the parent 19-
nor-1R,25-dihydroxyvitamin D3 can change completely
(and selectively) the biological potency of the analogues.
Thus, introduction of a 2â-hydroxy group into ring A of
2, which by itself has no calcemic activity,7b yielded a
vitamin D compound of high intestinal calcium trans-
port activity and no ability to mobilize calcium from
bone.9 An exomethylene substituent at C-2 has an
opposite effect, providing the analogue that very ef-
fectively increased bone calcium mobilization (serum
calcium) while being inactive in intestine. The present
results suggest that the 2-methylene- and 2R-methyl-
substituted 19-nor-1R,25-dihydroxyvitamin D3 com-
pounds in the 20S-series have very strong and prefer-
ential activity on bone, making them candidates for
treatment of bone diseases.
a
model 6000A solvent delivery system, a model 6 UK
Universal injector, a model 486 tunable absorbance detector,
and a differential R 401 refractometer. Microanalyses of
crystalline compounds were within (0.4% of the theoretical
values. THF was freshly distilled before use from sodium
benzophenone ketyl under argon.
The starting methyl quinicate derivative 15 was obtained
from commercial (-)-quinic acid as described previously.7
1
15: mp 82-82.5 °C (from hexane); H NMR (CDCl3) δ 0.098,
0.110, 0.142, and 0.159 (each 3H, each s, 4 × SiCH3), 0.896
and 0.911 (9H and 9H, each s, 2 × Si-t-Bu), 1.820 (1H, dd, J
) 13.1, 10.3 Hz), 2.02 (1H, ddd, J ) 14.3, 4.3, 2.4 Hz), 2.09
(1H, dd, J ) 14.3, 2.8 Hz), 2.19 (1H, ddd, J ) 13.1, 4.4, 2.4
Hz), 2.31 (1H, d, J ) 2.8 Hz, OH), 3.42 (1H, m; after D2O dd,
J ) 8.6, 2.6 Hz), 3.77 (3H, s), 4.12 (1H, m), 4.37 (1H, m), 4.53
(1H, br s, OH).
(3R,5R)-3,5-Bis[(ter t-bu tyldim eth ylsilyl)oxy]-1-h ydr oxy-
4-oxocycloh exa n eca r boxylic Acid Meth yl Ester (16). To
a stirred mixture of ruthenium(III) chloride hydrate (434 mg,
2.1 mmol) and sodium periodate (10.8 g, 50.6 mmol) in water
(42 mL) was added a solution of methyl quinicate 15 (6.09 g,
14 mmol) in CCl4/CH3CN (1:1, 64 mL). Vigorous stirring was
continued for 8 h. Few drops of 2-propanol were added; the
mixture was poured into water and extracted with chloroform.
The organic layer was washed with water, dried (MgSO4), and
evaporated to give a dark oily residue (ca. 5 g) which was
purified by flash chromatography. Elution with hexane/ethyl
The present study allows us also to conclude that the
axial orientation of the 1R-hydroxyl group in the vitamin
D molecule is essential for its biological potency.
Exp er im en ta l Section
Ch em istr y. Ultraviolet (UV) absorption spectra were
recorded with a Hitachi Model 60-100 UV-vis spectrometer
in the solvent noted. 1H nuclear magnetic resonance (NMR)

4670 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Sicinski et al.
acetate (8:2) gave pure, oily 4-ketone 16 (3.4 g, 56%): 1H NMR
(CDCl3) δ 0.054, 0.091, 0.127, and 0.132 (each 3H, each s, 4 ×
SiCH3), 0.908 and 0.913 (9H and 9H, each s, 2 × Si-t-Bu), 2.22
(1H, dd, J ) 13.2, 11.7 Hz), 2.28 (1H, ∼ dt, J ) 14.9, 3.6 Hz),
2.37 (1H, dd, J ) 14.9, 3.2 Hz), 2.55 (1H, ddd, J ) 13.2, 6.4,
3.4 Hz), 3.79 (3H, s), 4.41 (1H, t, J ∼ 3.5 Hz), 4.64 (1H, s, OH),
5.04 (1H, dd, J ) 11.7, 6.4 Hz); MS m/z (relative intensity) no
M+, 375 (M+ - t-Bu, 32), 357 (M+ - t-Bu - H2O, 47), 243 (31),
225 (57), 73 (100).
and benzene. The extract was washed with brine, dried
(MgSO4), and evaporated. An oily residue was dissolved in
hexane (1 mL) and applied on a Sep-Pak cartridge. Pure
4-methylenecyclohexanone derivative 19 (110 mg, 82%) was
eluted with hexane/ethyl acetate (95:5) as a colorless oil: 1H
NMR (CDCl3) δ 0.050 and 0.069 (6H and 6H, each s, 4 ×
SiCH3), 0.881 (18H, s, 2 × Si-t-Bu), 2.45 (2H, ddd, J ) 14.2,
6.9, 1.4 Hz), 2.64 (2H, ddd, J ) 14.2, 4.6, 1.4 Hz), 4.69 (2H,
dd, J ) 6.9, 4.6 Hz), 5.16 (2H, s); MS m/z (relative intensity)
no M+, 355 (M+ - Me, 3), 313 (M+ - t-Bu, 100), 73 (76).
(3R,5R)-3,5-Bis[(ter t-bu tyldim eth ylsilyl)oxy]-1-h ydr oxy-
4-m eth ylen ecycloh exa n eca r boxylic Acid Meth yl Ester
(17). To the methyltriphenylphoshonium bromide (2.813 g,
7.88 mmol) in anhydrous THF (32 mL) at 0 °C was added
dropwise n-BuLi (2.5 M in hexanes, 6.0 mL, 15 mmol) under
argon with stirring. Another portion of MePh3P+Br- (2.813
g, 7.88 mmol) was then added, and the solution was stirred at
0 °C for 10 min and at room temperature for 40 min. The
orange-red mixture was again cooled to 0 °C, and a solution
of 4-ketone 16 (1.558 g, 3.6 mmol) in anhydrous THF (16 + 2
mL) was siphoned to reaction flask during 20 min. The
reaction mixture was stirred at 0 °C for 1 h and at room
temperature for 3 h. The mixture was then carefully poured
into brine containing 1% HCl and extracted with ethyl acetate
and benzene. Organic extract was washed with diluted
NaHCO3 and brine, dried (MgSO4), and evaporated to give an
orange oily residue (ca. 2.6 g) which was purified by flash
chromatography. Elution with hexane/ethyl acetate (9:1) gave
pure 4-methylene compound 17 as a colorless oil (368 mg,
24%): 1H NMR (CDCl3) δ 0.078, 0.083, 0.092, and 0.115 (each
3H, each s, 4 × SiCH3), 0.889 and 0.920 (9H and 9H, each s,
2 × Si-t-Bu), 1.811 (1H, dd, J ) 12.6, 11.2 Hz), 2.10 (2H, m),
2.31 (1H, dd, J ) 12.6, 5.1 Hz), 3.76 (3H, s), 4.69 (1H, t, J )
3.1 Hz), 4.78 (1H, m), 4.96 (2H, m; after D2O 1H, br s), 5.17
(1H, t, J ) 1.9 Hz); MS m/z (relative intensity) no M+, 373
(M+ - t-Bu, 57), 355 (M+ - t-Bu - H2O, 13), 341 (19), 313
(25), 241 (33), 223 (37), 209 (56), 73 (100).
[(3′R,5′R)-3′,5′-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-1′-h y-
d r oxy-4′-m eth ylen ecycloh exyl]m eth a n ol (18). (a ) To a
stirred solution of the ester 17 (90 mg, 0.21 mmol) in
anhydrous THF (8 mL) was added lithium aluminum hydride
(60 mg, 1.6 mmol) at 0 °C under argon. The cooling bath was
removed after 1 h, and the stirring was continued at 6 °C for
12 h and at room temperature for 6 h. The excess of the
reagent was decomposed with saturated aqueous Na2SO4, and
the mixture was extracted with ethyl acetate and ether, dried
(MgSO4), and evaporated. Flash chromatography of the
residue with hexane/ethyl acetate (9:1) afforded unreacted
substrate (12 mg) and a pure, crystalline diol 18 (35 mg, 48%
based on recovered ester 17): 1H NMR (CDCl3 + D2O) δ 0.079,
0.091, 0.100, and 0.121 (each 3H, each s, 4 × SiCH3), 0.895
and 0.927 (9H and 9H, each s, 2 × Si-t-Bu), 1.339 (1H, t, J ∼
12 Hz), 1.510 (1H, dd, J ) 14.3, 2.7 Hz), 2.10 (2H, m), 3.29
and 3.40 (1H and 1H, each d, J ) 11.0 Hz), 4.66 (1H, t, J ∼
2.8 Hz), 4.78 (1H, m), 4.92 (1H, t, J ) 1.7 Hz), 5.13 (1H, t, J
) 2.0 Hz); MS m/z (relative intensity) no M+, 345 (M+ - t-Bu,
8), 327 (M+ - t-Bu - H2O, 22), 213 (28), 195 (11), 73 (100).
Anal. (C20H42O4Si2) C, H.
[(3′R,5′R)-3′,5′-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-4′-m e-
th ylen ecycloh exylid en e]a cetic Acid Meth yl Ester (20).
To a solution of diisopropylamine (37 µL, 0.28 mmol) in
anhydrous THF (200 µL) was added n-BuLi (2.5 M in hexanes,
113 µL, 0.28 mmol) under argon at -78 °C with stirring, and
methyl (trimethylsilyl)acetate (46 µL, 0.28 mmol) was then
added. After 15 min, the keto compound 19 (49 mg, 0.132
mmol) in anhydrous THF (200 + 80 µL) was added dropwise.
The solution was stirred at -78 °C for 2 h, and the reaction
mixture was quenched with saturated NH4Cl, poured into
brine and extracted with ether and benzene. The combined
extracts were washed with brine, dried (MgSO4), and evapo-
rated. The residue was dissolved in hexane (1 mL) and applied
on a Sep-Pak cartridge. Elution with hexane and hexane/ethyl
acetate (98:2) gave a pure allylic ester 20 (50 mg, 89%) as a
colorless oil: 1H NMR (CDCl3) δ 0.039, 0.064, and 0.076 (6H,
3H, and 3H, each s, 4 × SiCH3), 0.864 and 0.884 (9H and 9H,
each s, 2 × Si-t-Bu), 2.26 (1H, dd, J ) 12.8, 7.4 Hz), 2.47 (1H,
dd, J ) 12.8, 4.2 Hz), 2.98 (1H, dd, J ) 13.3, 4.0 Hz), 3.06
(1H, dd, J ) 13.3, 6.6 Hz), 3.69 (3H, s), 4.48 (2H, m), 4.99 (2H,
s), 5.74 (1H, s); MS m/z (relative intensity) 426 (M+, 2), 441
(M+ - Me, 4), 369 (M+ - t-Bu, 100), 263 (69).
2-[(3′R,5′R)-3′,5′-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-4′-
m eth ylen ecycloh exylid en e]eth a n ol (21). Diisobutylalu-
minum hydride (1.5 M in toluene, 1.6 mL, 2.4 mmol) was
slowly added to a stirred solution of the allylic ester 20 (143
mg, 0.33 mmol) in toluene/methylene chloride (2:1, 5.7 mL) at
-78 °C under argon. Stirring was continued at -78 °C for 1
h and at -46 °C (cyclohexanone/dry ice bath) for 25 min. The
mixture was quenched by the slow addition of potassium
sodium tartrate (2 N, 3 mL), aqueous HCl (2 N, 3 mL), and
H2O (12 mL) and then diluted with methylene chloride (12
mL) and extracted with ether and benzene. The organic layers
were washed with diluted (ca. 1%) HCl and brine, dried
(MgSO4), and evaporated. The residue was purified by flash
chromatography. Elution with hexane/ethyl acetate (9:1) gave
crystalline allylic alcohol 21 (130 mg, 97%): 1H NMR (CDCl3)
δ 0.038, 0.050, and 0.075 (3H, 3H, and 6H, each s, 4 × SiCH3),
0.876 and 0.904 (9H and 9H, each s, 2 × Si-t-Bu), 2.12 (1H,
dd, J ) 12.3, 8.8 Hz), 2.23 (1H, dd, J ) 13.3, 2.7 Hz), 2.45
(1H, dd, J ) 12.3, 4.8 Hz), 2.51 (1H, dd, J ) 13.3, 5.4 Hz),
4.04 (1H, m; after D2O dd, J ) 12.0, 7.0 Hz), 4.17 (1H, m; after
D2O dd, J ) 12.0, 7.4 Hz), 4.38 (1H, m), 4.49 (1H, m), 4.95
(1H, br s), 5.05 (1H, t, J ) 1.7 Hz), 5.69 (1H, ∼t, J ) 7.2 Hz);
MS m/z (relative intensity) 398 (M+, 2), 383 (M+ - Me, 2), 365
(M+ - Me - H2O, 4), 341 (M+ - t-Bu, 78), 323 (M+ - t-Bu -
H2O, 10), 73 (100). Anal. (C21H42O3Si2) C, H.
(b) Diisobutylaluminum hydride (1.5 M in toluene, 2.0 mL,
3 mmol) was added to a solution of the ester 17 (215 mg, 0.5
mmol) in anhydrous ether (3 mL) at -78 °C under argon. The
mixture was stirred at -78 °C for 3 h and at -24 °C for 1.5 h,
diluted with ether (10 mL), and quenched by the slow addition
of 2 N potassium sodium tartrate. The solution was warmed
to room temperature, stirred for 15 min, and then poured into
brine and extracted with ethyl acetate and ether. Organic
extracts were washed with diluted (ca. 1%) HCl and brine,
dried (MgSO4), and evaporated. The crystalline residue was
purified by flash chromatography. Elution with hexane/ethyl
acetate (9:1) gave crystalline diol 18 (43 mg, 24%).
(3R,5R)-3,5-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-4-m eth yl-
en ecycloh exa n on e (19). Sodium periodate-saturated water
(2.2 mL) was added to a solution of the diol 18 (146 mg, 0.36
mmol) in methanol (9 mL) at 0 °C. The solution was stirred
at 0 °C for 1 h, poured into brine, and extracted with ether
[2-[(3′R,5′R)-3′,5′-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-4′-
m eth ylen ecycloh exyliden e]eth yl]diph en ylph osph in e Ox-
id e (22). To the allylic alcohol 21 (105 mg, 0.263 mmol) in
anhydrous THF (2.4 mL) was added n-BuLi (2.5 M in hexanes,
105 µL, 0.263 mmol) under argon at 0 °C. Freshly recrystal-
lized tosyl chloride (50.4 mg, 0.264 mmol) was dissolved in
anhydrous THF (480 µL) and added to the allylic alcohol-
BuLi solution. The mixture was stirred at 0 °C for 5 min and
set aside at 0 °C. In another dry flask with air replaced by
argon, n-BuLi (2.5 M in hexanes, 210 µL, 0.525 mmol) was
added to Ph2PH (93 µL, 0.534 mmol) in anhydrous THF (750
µL) at 0 °C with stirring. The red solution was siphoned under
argon pressure to the solution of tosylate until the orange color
persisted (ca. one-half of the solution was added). The
resulting mixture was stirred for an additional 30 min at 0 °C
and quenched by addition of H2O (30 µL). Solvents were
evaporated under reduced pressure, and the residue was

New 1R,25-(OH)2D3 with High Biological Activity
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23 4671
redissolved in methylene chloride (2.4 mL) and stirred with
10% H2O2 at 0 °C for 1 h. The organic layer was separated,
washed with cold aqueous sodium sulfite and H2O, dried
(MgSO4), and evaporated. The residue was subjected to flash
chromatography. Elution with benzene/ethyl acetate (6:4)
gave semicrystalline phosphine oxide 22 (134 mg, 87%): 1H
NMR (CDCl3) δ 0.002, 0.011, and 0.019 (3H, 3H, and 6H, each
s, 4 × SiCH3), 0.855 and 0.860 (9H and 9H, each s, 2 × Si-t-
Bu), 2.0-2.1 (3H, br m), 2.34 (1H, m), 3.08 (1H, m), 3.19 (1H,
m), 4.34 (2H, m), 4.90 and 4.94 (1H and 1H, each s,), 5.35 (1H,
∼q, J ) 7.4 Hz), 7.46 (4H, m), 7.52 (2H, m), 7.72 (4H, m); MS
(56 mg, 0.2 mmol) and imidazole (65 mg, 0.95 mmol) in
anhydrous DMF (1.2 mL) was treated with triethylsilyl
chloride (95 µL, 0.56 mmol), and the mixture was stirred at
room temperature under argon for 4 h. Ethyl acetate was
added and water, and the organic layer was separated. The
ethyl acetate layer was washed with water and brine, dried
(MgSO4), and evaporated. The residue was passed through a
silica Sep-Pak cartridge in hexane/ethyl acetate (9:1) and, after
evaporation, purified by HPLC (9.4-mm × 25-cm Zorbax-Sil
column, 4 mL/min) using hexane/ethyl acetate (9:1) solvent
system. Pure protected hydroxy ketone 23b (55 mg, 70%) was
eluted at RV 35 mL as a colorless oil: 1H NMR (CDCl3) δ 0.566
(6H, q, J ) 7.9 Hz, 3 × SiCH2), 0.638 (3H, s, 18-H3), 0.859
(3H, d, J ) 6.0 Hz, 21-H3), 0.947 (9H, t, J ) 7.9 Hz, 3 ×
SiCH2CH3), 1.196 (6H, s, 26- and 27-H3), 2.45 (1H, dd, J )
11.4, 7.5 Hz, 14R-H).
(20S)-1r,25-Dih yd r oxy-2-m eth ylen e-19-n or vita m in D3
(6b). Vitamin 6b was prepared in the same way as 6a , except
from the corresponding 23b. The protected compound 24b was
isolated by flash chromatography in 79% yield. For analytical
purposes a sample of vitamin 24b was further purified by
HPLC (6.2-mm × 25-cm Zorbax-Sil column, 4 mL/min) using
hexane/ethyl acetate (99.9:0.1) solvent system.
Protected vitamin 24b was hydrolyzed as described for 24a ,
and the product was purified by HPLC (6.2-mm × 25-cm
Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (9:1)
solvent system. Analytically pure 2-methylene-19-norvitamin
6b (99% yield) was collected at RV 28 mL as a white solid:
UV (in EtOH) λmax 243.5, 252.5, 262.5 nm; 1H NMR (CDCl3) δ
0.551 (3H, s, 18-H3), 0.858 (3H, d, J ) 6.6 Hz, 21-H3), 1.215
(6H, s, 26- and 27-H3), 1.95-2.04 (2H, m), 2.30 (1H, dd, J )
13.3, 8.4 Hz, 10R-H), 2.33 (1H, dd, J ) 13.4, 6.3 Hz, 4â-H),
2.58 (1H, dd, J ) 13.4, 3.7 Hz, 4R-H), 2.83 (1H, br d, J ) 13.7
Hz, 9â-H), 2.85 (1H, dd, J ) 13.3, 4.5 Hz, 10â-H), 4.49 (2H,
m, 1â- and 3R-H), 5.09 and 5.11 (1H and 1H, each s, dCH2),
5.89 and 6.36 (1H and 1H, each d, J ) 11.3 Hz, 7- and 6-H);
MS m/z (relative intensity) 416 (M+, 100), 398 (26), 380 (13),
366 (21), 313 (31); exact mass calcd for C27H44O3 416.3290,
found 416.3275.
1r,25-Dih yd r oxy-2r- a n d 1r,25-Dih yd r oxy-2â-m eth yl-
19-n or vita m in D3 (7a a n d 8a ). Tris(triphenylphosphine)-
rhodium(I) chloride (23 mg, 25 µmol) was added to dry benzene
(25 mL) presaturated with hydrogen. The mixture was stirred
at room temperature until a homogeneous solution was formed
(ca. 45 min). A solution of vitamin 6a (10 mg, 24 µmol) in dry
benzene (5 mL) was then added, and the reaction was allowed
to proceed under a continuous stream of hydrogen for 3 h.
Benzene was removed under vacuum; the residue was redis-
solved in hexane/ethyl acetate (1:1, 4 mL) and applied on
Waters silica Sep-Pak column (vac 12 cm3). A mixture of
2-methyl vitamins was eluted with the same solvent system
(20 mL). The compounds were further purified by HPLC (6.2-
mm × 25-cm Zorbax-Sil column, 4 mL/min) using hexane/2-
propanol (85:15) solvent system. The mixture (ca. 1:1) of
2-methyl-19-norvitamins 7a and 8a (8.0 mg, 80%) gave a single
peak at RV 17 mL. Separation of both epimers was achieved
by reversed-phase HPLC (10-mm × 25-cm Zorbax-ODS col-
umn, 4 mL/min) using methanol/water (85:15) solvent system.
2â-Methyl vitamin 8a (3.5 mg, 35%) was collected at RV 41
mL and its 2R-epimer 7a (3.4 mg, 34%) at RV 46 mL.
(20S)-1r,25-Dih yd r oxy-2r- a n d (20S)-1r,25-Dih yd r oxy-
2â-m eth yl-19-n or vita m in D3 (7b a n d 8b). Vitamins 7b and
8b were obtained by hydrogenation of 6b, performed analo-
gously to the process described above for 20R-isomer 6a . The
hydrogenated compounds were first purified by HPLC (6.2-
mm × 25-cm Zorbax-Sil column, 4 mL/min) using hexane/2-
propanol (85:15) solvent system. The mixture of 2-methyl-19-
norvitamins 7b and 8b (ca. 1:1, 45%) gave a single peak at RV
16.5 mL. Separation of both epimers was achieved by reversed-
phase HPLC (10-mm × 25-cm Zorbax-ODS column, 4 mL/min)
using methanol/water (85:15) solvent system. 2â-Methyl
vitamin 8b (16%) was collected at RV 36 mL and its 2R-epimer
7b (20%) at RV 45 mL.
m/z (relative intensity) no M+, 581 (M+ - 1, 1), 567 (M+
-
Me, 3), 525 (M+ - t-Bu, 100), 450 (10), 393 (48). Anal.
(C33H51O3PSi2) C, H.
1r,25-Dih yd r oxy-2-m eth ylen e-19-n or vita m in D3 (6a ).
To a solution of phosphine oxide 22 (33.1 mg, 56.8 µmol) in
anhydrous THF (450 µL) at 0 °C was slowly added n-BuLi (2.5
M in hexanes, 23 µL, 57.5 µmol) under argon with stirring.
The solution turned deep orange. The mixture was cooled to
-78 °C, and a precooled (-78 °C) solution of protected hydroxy
ketone 23a (9.0 mg, 22.8 µmol; prepared according to a
published procedure9) in anhydrous THF (200 + 100 µL) was
slowly added. The mixture was stirred under argon at -78
°C for 1 h and at 0 °C for 18 h. Ethyl acetate was added, and
the organic phase was washed with brine, dried (MgSO4), and
evaporated. The residue was dissolved in hexane, applied on
a silica Sep-Pak cartridge, and washed with hexane/ethyl
acetate (99.5:0.5, 20 mL) to give 19-norvitamin derivative 24a
(13.5 mg, 78%). The Sep-Pak was then washed with hexane/
ethyl acetate (96:4, 10 mL) to recover some unchanged C,D-
ring ketone 23a (2 mg) and with ethyl acetate (10 mL) to
recover diphenylphosphine oxide (20 mg). For analytical
purpose a sample of protected vitamin 24a was further purified
by HPLC (6.2-mm × 25-cm Zorbax-Sil column, 4 mL/min)
using hexane/ethyl acetate (99.9:0.1) solvent system. Pure
compound 24a was eluted at RV 26 mL as a colorless oil: UV
1
(in hexane) λmax 244, 253, 263 nm; H NMR (CDCl3) δ 0.025,
0.049, 0.066, and 0.080 (each 3H, each s, 4 × SiCH3), 0.546
(3H, s, 18-H3), 0.565 (6H, q, J ) 7.9 Hz, 3 × SiCH2), 0.864
and 0.896 (9H and 9H, each s, 2 × Si-t-Bu), 0.931 (3H, d, J )
6.0 Hz, 21-H3), 0.947 (9H, t, J ) 7.9 Hz, 3 × SiCH2CH3), 1.188
(6H, br s, 26- and 27-H3), 2.00 (2H, m), 2.18 (1H, dd, J ) 12.5,
8.5 Hz, 4â-H), 2.33 (1H, dd, J ) 13.1, 2.9 Hz, 10â-H), 2.46 (1H,
dd, J ) 12.5, 4.5 Hz, 4R-H), 2.52 (1H, dd, J ) 13.1, 5.8 Hz,
10R-H), 2.82 (1H, br d, J ) 12 Hz, 9â-H), 4.43 (2H, m, 1â- and
3R-H), 4.92 and 4.97 (1H and 1H, each s, dCH2), 5.84 and 6.22
(1H and 1H, each d, J ) 11.0 Hz, 7- and 6-H); MS m/z (relative
intensity) 758 (M+, 17), 729 (M+ - Et, 6), 701 (M+ - t-Bu, 4),
626 (100), 494 (23), 366 (50), 73 (92).
Protected vitamin 24a (4.3 mg) was dissolved in benzene
(150 µL), and the resin (AG 50W-X4, 60 mg; prewashed with
methanol) in methanol (800 µL) was added. The mixture was
stirred at room temperature under argon for 17 h, diluted with
ethyl acetate/ether (1:1, 4 mL), and decanted. The resin was
washed with ether (8 mL), and the combined organic phases
were washed with brine and saturated NaHCO3, dried (Mg-
SO4), and evaporated. The residue was purified by HPLC (6.2-
mm × 25-cm Zorbax-Sil column, 4 mL/min) using hexane/2-
propanol (9:1) solvent system. Analytically pure 2-methylene-
19-norvitamin 6a (2.3 mg, 97%) was collected at RV 29 mL
(1R,25-dihydroxyvitamin D3 was eluted at RV 52 mL in the
same system) as a white solid: UV (in EtOH) λmax 243.5, 252,
262.5 nm; 1H NMR (CDCl3) δ 0.552 (3H, s, 18-H3), 0.941 (3H,
d, J ) 6.4 Hz, 21-H3), 1.222 (6H, s, 26- and 27-H3), 2.01 (2H,
m), 2.29 (1H, dd, J ) 13.3, 8.4 Hz, 10R-H), 2.33 (1H, dd, J )
13.4, 6.0 Hz, 4â-H), 2.58 (1H, dd, J ) 13.4, 3.9 Hz, 4R-H), 2.82
(1H, br d, J ) 12 Hz, 9â-H), 2.86 (1H, dd, J ) 13.3, 4.6 Hz,
10â-H), 4.49 (2H, m, 1â- and 3R-H), 5.10 and 5.11 (1H and
1H, each s, dCH2), 5.89 and 6.37 (1H and 1H, each d, J )
11.3 Hz, 7- and 6-H); MS m/z (relative intensity) 416 (M+, 83),
398 (25), 384 (31), 380 (14), 351 (20), 313 (100); exact mass
calcd for C27H44O3 416.3290, found 416.3279.
(20S)-25-[(Tr iet h ylsilyl)oxy]-d es-A,B-ch olest a n -8-on e
(23b). A solution of (20S)-25-hydroxy Grundmann’s ketone14
1r,25-Dih ydr oxy-2r- an d 1r,25-Dih ydr oxy-2â-(h ydr oxy-
m eth yl)-19-n or vita m in D3 (9a a n d 10a ). 9-Borabicyclo-

4672 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 23
Sicinski et al.
[3.3.1]nonane (0.5 M in THF, 60 µL, 30 µmol) was added to a
solution of vitamin 6a (1.25 mg, 3 µmol) in anhydrous THF
(50 µL) at room temperature (evolution of hydrogen was
observed). After 3 h of stirring, the mixture was quenched
with methanol (20 µL), stirred for 15 min at room temperature,
cooled to 0 °C, and treated successively with 6 M NaOH (10
µL, 60 µmol) and 30% H2O2 (10 µL). The mixture was heated
for 1 h at 55 °C and cooled, benzene and brine were added,
and the organic phase was separated, dried, and evaporated.
The crystalline residue was dissolved in ether (0.5 mL) and
kept in a freezer overnight. The ether solution was carefully
removed from the precipitated crystals of cyclooctanediol and
evaporated. Separation of the residue was achieved by HPLC
(6.2-mm × 25-cm Zorbax-Sil column, 4 mL/min) using hexane/
2-propanol (85:15) solvent system. Traces of unreacted sub-
strate 6a were eluted at RV 16 mL, whereas isomeric 2-hy-
droxymethyl vitamins 9a and 10a were collected at RV 33 and
40 mL, respectively. Further purification of both products by
reversed-phase HPLC (10-mm × 25-cm Zorbax-ODS column,
4 mL/min) using methanol/water (9:1) solvent system afforded
analytically pure vitamin 9a (0.14 mg, 11%) and its 2â-isomer
10a (0.31 mg, 24%) collected at RV 26 and 23 mL, respectively.
spectrometer model 3110. Intestinal calcium transport was
determined by the everted intestinal sac method using the
proximal 10 cm of intestine as described earlier.32 Statistical
analysis was by the Student’s t-test.34 Intestinal calcium
transport is expressed as serosal:mucosal ratio of calcium in
the sac to the calcium in the final incubation medium, or S/M.
Bone calcium mobilization represents the rise in serum
calcium of the rats maintained on a very-low-calcium diet. In
that measurement, the rise in serum calcium must arise from
bone and hence is a determination of bone calcium mobiliza-
tion.
2. Mea su r em en t of Cellu la r Differ en tia tion . Human
leukemia HL-60 cells, originally obtained from ATTC, were
plated at 2 × 105 cells/plate and incubated in Eagle’s modified
medium as described previously.4d The compounds tested were
added in the indicated concentrations in 0.05 mL of ethanol
so that the ethanol concentration never exceeded 1%. The
incubation was carried out for 4 days, and at the end of the
fourth day, superoxide production was measured by nitro blue
tetrazolium (NBT) reduction. The number of cells containing
intracellular black-blue formazan deposits was determined by
light microscopy using a hemacytometer. At least 200 cells
were counted in duplicate per determination. Percentage
differentiation represents percentage cells providing NBT
reduction appearance. The results were plotted on semilog
paper, and relative differentiation activities of the analogues
were determined by comparison of the compound concentra-
tions capable of inducing 50% maturation according to the
assay. This method is described in detail elsewhere.4d The
experiment was repeated three times, and the results are
reported as the mean ( SD.
3. Mea su r em en t of Bin d in g to th e P or cin e In testin a l
Vita m in D Recep tor . Porcine intestinal nuclear extract was
prepared as described earlier.35 It was diluted, and 0.1 mg of
protein (200 fmol of binding activity) in 100 µL was used in
each tube; 10 000 cpm of 1R,25-(OH)2[26,27-3H]D3 was added
in 2.0 µL of ethanol. To this was added either standard
radioinert 1R,25-(OH)2D3 at various concentrations or the
indicated analogue at various concentrations in 5 µL of
ethanol. The mixture was incubated at room temperature for
4 h on a shaker and then 100 µL of hydroxyapatite (50% slurry)
added. The sample was vortexed at 5-min intervals for 15 min
on ice. The hydroxyapatite was then washed three times by
adding 0.5 mL of TE 5% Triton ×100, centrifuging at 200g for
5 min, and aspirating the supernatant. The radioactivity
bound to the hydroxyapatite was determined by liquid scintil-
lation counting in Bio-Safe II scintillation fluid. The values
are plotted versus concentration of analogue or standard.
Each value represents triplicate values. The displacement
experiments were carried out in triplicate on two different
occasions.
(20S)-1r,25-Dih yd r oxy-2r- a n d (20S)-1r,25-Dih yd r oxy-
2â-(h yd r oxym eth yl)-19-n or vita m in D3 (9b a n d 10b). The
hydroboration of 20S-vitamin 6b and subsequent oxidation of
the organoborane adduct were performed using the procedure
analogous to that described above for 20R-epimer 6a . The
reaction products were separated by HPLC (6.2-mm × 25-cm
Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (87.5:
12.5) solvent system, and the isomeric 2-hydroxymethyl vita-
mins 9b and 10b were collected at RV 40 and 47 mL,
respectively. Further purification of both products by reversed-
phase HPLC (10-mm × 25-cm Zorbax-ODS column, 4 mL/min)
using methanol/water (9:1) solvent system afforded analyti-
cally pure vitamin 9b (9%) and its 2â-isomer 10b (26%)
collected at RV 25 and 22 mL, respectively.
(10S)- a n d (10R)-1r,19,25-Tr ih yd r oxy-10,19-d ih yd r o-
vita m in D3 (13 a n d 14). The hydroboration of 1R,25-(OH)2D3
(1) and subsequent oxidation of the borane adduct were
performed using the procedure analogous to that described
above for vitamin 6a . The reaction products were subjected
to flash chromatography. Elution with ethyl acetate resulted
in separation of both isomers: vitamins 13 and 14 were
gradually eluted from the column. Further purification of
products by straight-phase HPLC (6.2-mm × 25-cm Zorbax-
Sil column, 4 mL/min) using hexane/2-propanol (8:2) solvent
system (both isomers collected at RV ca. 33 mL) and reversed-
phase HPLC (10-mm × 25-cm Zorbax-Sil column, 4 mL/min)
using methanol/water (9:1) solvent system (the isomers eluted
at RV ca. 19 mL) provided the analytically pure, crystalline
vitamins 13 (12%) and 14 (9%).
Biologica l Stu d ies. 1. Mea su r em en t of In testin a l
Ca lciu m Tr a n sp or t a n d Bon e Ca lciu m Mobiliza tion .
Twenty-day-old Weanling male rats from the low-vitamin D
colony were purchased from the Sprague-Dawley Co. (India-
napolis, IN) and fed the vitamin D-deficient diet,32 containing
0.47% calcium and 0.3% phosphorus, for 1 week. They were
then switched to the reduced calcium diet (0.02% Ca) for an
additional 2 weeks. These animals have no detectable levels
of 25-OH-D3 or 1R,25-(OH)2D3 in their plasma as measured
by methods described previously.33 For this first experiment,
the indicated rats received a single intravenous dose of the
indicated compound in 0.05 mL of ethanol (data not shown).
In the other experiment, the rats were given the indicated
doses of compounds in 0.1 mL of (95:5) 1,2-propanediol/ethanol
by intraperitoneal injection each day for 7 days. In the first
experiment, the rats were euthanized at various times after
the dose (data not shown). In the second experiment, they
were sacrificed 24 h after the last dose. The rats were
sacrificed under ether anesthesia by decapitation; their blood
and intestines were collected and used immediately to deter-
mine calcium transport activity and serum calcium concentra-
tion. Calcium was measured in the presence of 0.1% lantha-
nium chloride by means of a Perkin-Elmer atomic absorption
Ack n ow led gm en t. We acknowledge the assistance
of Rowland Randall in recording the mass spectra. The
work was supported in part by program project Grant
DK14881 from the National Institutes of Health and
the Wisconsin Alumni Research Foundation.
Su p p or tin g In for m a tion Ava ila ble: Spectral data of the
vitamin D compounds 7a ,b-10a ,b, 13, 14, and 24b; figures
with the competitive binding curves derived from the binding
assay of the vitamin D analogues 6a ,b-10a ,b; and figures with
dose-response curves derived from the cellular differentiation
assay performed for the same compounds (12 pages). Ordering
information is given on any current masthead page.
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J M9802618