2-Ethyl and 2-ethylidene analogues of 1α,25-dihydroxy-19-norvitamin D3: Synthesis, conformational analysis, biological activities, and docking to the modeled rVDR ligand binding domain

Article abstract of DOI:10.1021/jm020007m

Novel 19-nor analogues of 1α,25-dihydroxyvitamin D₃ were prepared and substituted at C-2 with an ethylidene group. The synthetic pathway was via Wittig - Horner coupling of the corresponding A-ring phosphine oxides with the protected 25-hydroxy

Full text of DOI:10.1021/jm020007m

3366
J . Med. Chem. 2002, 45, 3366-3380
2-Eth yl a n d 2-Eth ylid en e An a logu es of 1r,25-Dih yd r oxy-19-n or vita m in D3:
Syn th esis, Con for m a tion a l An a lysis, Biologica l Activities, a n d Dock in g to th e
Mod eled r VDR Liga n d Bin d in g Dom a in
Rafal R. Sicinski,†,§ Piotr Rotkiewicz,‡ Andrzej Kolinski,‡ Wanda Sicinska,† J ean M. Prahl,†
Connie M. Smith,† and Hector F. DeLuca*,†
Department of Biochemistry, University of WisconsinsMadison, 433 Babcock Drive, Madison, Wisconsin 53706, and
Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
Received J anuary 7, 2002
Novel 19-nor analogues of 1R,25-dihydroxyvitamin D3 were prepared and substituted at C-2
with an ethylidene group. The synthetic pathway was via Wittig-Horner coupling of the
corresponding A-ring phosphine oxides with the protected 25-hydroxy Grundmann’s ketones.
Selective catalytic hydrogenation of 2-ethylidene analogues provided the 2R- and 2â-ethyl
compounds. The 2-ethylidene-19-nor compounds with a methyl group from the ethylidene moiety
in a trans relationship to the C(6)-C(7) bond (E-isomers) were more potent than the
corresponding Z-isomers and the natural hormone in binding to the vitamin D receptor. Both
geometrical isomers (E and Z) of (20S)-2-ethylidene-19-norvitamin D3 and both 2R-ethyl-19-
norvitamins (in the 20R- and 20S-series) have much higher HL-60 differentiation activity than
does 1R,25-(OH)2D3. Both E-isomers (20R and 20S) of 2-ethylidene vitamins are characterized
by very high calcemic activity in rats. The three-dimensional structure model of the rat vitamin
D receptor and the computational docking of four synthesized (20R)-19-norvitamin D3 analogues
into its binding pocket are also reported.
In tr od u ction
with ethyl (6a ,b and 7a ,b) and ethylidene (8a ,b and
9a ,b) groups. We also performed docking molecular
simulations with the four vitamins from the 20R-series
(6a -9a ) using the computational model of the full-
length ligand binding domain of the rat vitamin D
receptor (rVDR-LBD).
The discovery of the hormonally active form of vita-
min D3, 1R,25-dihydroxyvitamin D3 (1R,25-(OH)2D3,
calcitriol, 1, Figure 1), has greatly stimulated research
into its physiology and chemistry.1,2 It has been estab-
lished that 1 not only regulates the mineral metabolism
in animals and humans3 but also affects the human
immune system and exerts potent effects upon cell
proliferation and cellular differentiation.4 Therefore, the
study of the chemistry of vitamin D has been recently
focused on the design and synthesis of such analogues
which can exert selective biological actions.5 In our
continuing investigation of the structure-activity rela-
tionship of the vitamin D molecule, we prepared an
analogue of the natural hormone 1, 1R,25-dihydroxy-2-
methylene-19-norvitamin D3 (2a ),6 in which the exocy-
clic methylene group is transposed, in comparison with
1, from C-10 to C-2. Also, the 2R-methyl analogue 4a
was obtained by a selective hydrogenation of 2. Both
analogues, being formally derivatives of 19-nor-1R,25-
(OH)2D3 (3a ) synthesized by us previously,7 were char-
acterized by significant biological potency, enhanced
especially in their isomers in the 20S-series (2b, 4b).6
As a continuation of our search for biologically active
vitamin D compounds possessing A-ring conformational
equilibrium shifted to one particular chair form, we
prepared novel 19-nor analogues of 1 substituted at C-2
Resu lts a n d Discu ssion
Ch em istr y. The strategy of synthesis was based on
the Lythgoe-type Wittig-Horner coupling approach8
which was successfully used by us in the preparation
of other 2-substituted 19-norvitamins.6,9 Availability of
the corresponding protected 25-hydroxy Grundmann’s
ketones with “natural” (19a )9 and inverted (19b )6
configurations at C-20 directed our attention to the
preparation of the phosphine oxide A-ring synthons 17
and 18 (Scheme 1).
The hydroxycyclohexanone compound 10 was synthe-
sized in 4 steps (25% yield) from commercially available
(1R,3R,4S,5R)-(-)-quinic acid.10 Catalytic oxidation with
ruthenium tetroxide afforded cyclohexanedione deriva-
tive 11 in 65% yield. Due to a considerable difference
in the steric hindrance between the two carbonyl groups,
it was possible to achieve selective Peterson olefination
with methyl(trimethylsilyl)acetate providing allylic es-
ter 12 in 67% yield. However, the following Wittig
reaction with a ylide generated from ethyltriphenylphos-
phonium bromide and n-BuLi proved to be much less
efficient. Our attempts to improve the yield (18%) by
changing reaction conditions and bases used for the
ylide preparation (for example, potassium and sodium
* To whom correspondence should be addressed: Department of
Biochemistry, University of WisconsinsMadison, 433 Babcock Drive,
Madison, WI 53706. Telephone: 608-262-1620. Fax: 608-262-7122.
E-mail: deluca@biochem.wisc.edu.
† University of Wisconsin-Madison.
1
bis(trimethylsilyl)amides) were not successful. The H
‡ University of Warsaw.
NMR spectrum of the resulting mixture of 4′-ethylidene
compounds 13 and 14 indicated a presence of two
§ Present address: Department of Chemistry, University of Warsaw,
ul. Pasteura 1, 02-093 Warsaw, Poland.
10.1021/jm020007m CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/28/2002

Analogues of 1R,25-Dihydroxy-19-norvitamin D3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3367
F igu r e 1. Chemical structure of 1R,25-dihydroxyvitamin D3 (calcitriol, 1) and its analogues.
equatorial proton at C-5′ and, therefore, in E relation-
geometrical isomers (1:1.7 ratio) differing in the orienta-
tion of a carbomethoxy group and a methyl group from
the ethylidene moiety (both groups are considered
substituents of terminal carbon atoms in a 1,4-dimeth-
ylenecyclohexane fragment). Although the spectrum was
characterized by good proton dispersion, the assignment
of their structures was performed after the following
DIBALH reduction step due to difficulties with chro-
matographic separation of the isomers. Allylic alcohols
15 and 16 (80% yield) were easily separated by prepara-
tive HPLC, and their configurations as well as preferred
conformations were unequivocally established by 1H
NOE difference spectroscopy and spin decoupling ex-
periments. Analysis of the proton coupling network
supported the expected strong preference of chair
conformers possessing an axial orientation of this
OTBDMS group to which the methyl group from 4′-
ethylidene moiety is directed. For example, in the major
isomer (Figure 2b), a signal of an equatorial proton at
C-6′ (δ 2.50) was assigned by a NOE experiment
involving vinylic 2-H (δ 5.73). The coupling constant of
its geminal partner resonating at δ 2.02 with the vicinal
proton at C-5′ (ca. 11 Hz) is typical for protons in axial/
axial orientation and clearly indicates a significant bias
toward one chair-like cyclohexane ring conformation
with the 3′- and 5′-OTBDMS groups in axial and
equatorial orientation, respectively. Two NOE experi-
ments involving the irradiation of axial 5′R-H and
equatorial 3′â-H show a 2.6% enhancement of a vinylic
proton (δ 5.59) from the ethylidene unit and a 12.2%
enhancement of the methyl signal (δ 1.70), respectively,
supporting a Z relationship of the latter with the
1-hydroxymethyl group (16). The coupling constants and
the observed NOEs in the minor isomer presented in
Figure 2a demonstrate that the methyl substituent of
the ethylidene moiety is in close proximity to the
ship to 1-CH2OH (15). Each of the isomeric allylic
alcohols 15 and 16 was then transformed to the desired
A-ring phosphine oxide synthons 17 and 18 using an
efficient (ca. 60%) three-step procedure of tosylation,
reaction with a diphenylphosphine anion, and oxidation
with hydrogen peroxide. The subsequent Wittig-Horner
reaction of the conjugate base of 17 with the protected
25-hydroxy Grundmann’s ketone 19a produced 19-
norvitamin D3 compound 20a in excellent yield (91%),
but the yield of an analogous coupling of the isomeric
phosphine oxide 18 was very low (13%).
A striking difference in the reactivity of the isomeric
phosphine oxides in Wittig-Horner coupling with 19a
was nicely demonstrated when the 1:2 mixture of 17
and 18 was used. This resulted in the formation of
practically pure 19-norvitamin D3 compound 20a de-
rived from the minor E-isomer! A possible explanation
of this intriguing observation can be obtained from
the consideration of a steric hindrance in the corre-
sponding intermediates formed in the first step of the
Wittig-Horner reaction. Most likely, an attack of the
lithium phosphinoxy carbanion, produced from 18 and
n-BuLi, on C-8 takes place from the R-side of the
Grundmann’s ketone molecule and results in the 8â-
hydroxy adduct. The configuration of the ethylidene
substituent forces the 1R-OTBDMS group to adopt an
axial orientation, and this bulky substituent, interacting
with substituents of the phosphorus atom, restricts
rotation around the C(7)-C(8) bond, destabilizing the
syn-periplanar conformation of the P-C(7)-C(8)-O
fragment (Figure 3) required for a syn-elimination of
diphenylphosphinic acid11,12 and the formation of a 7E-
double bond. On the basis of the literature data indicat-
ing reversibility of the first step of Wittig reactions
involving phosphine oxides,11 the low yield of the diene

3368 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Sicinski et al.
1
F igu r e 2. Preferred conformations of the synthesized allylic alcohols 15 (a) and 16 (b). The most informative NOEs and H-1H
coupling constants are given.
Sch em e 1
21a and recovery of the unchanged phosphine oxide 18
from the coupling of the isomeric phosphine oxide 17,
can be expected. In the case of an intermediate resulting
A-ring substituents do not exert an additional steric

Analogues of 1R,25-Dihydroxy-19-norvitamin D3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3369
F igu r e 3. Stereoviews of the intermediates of the Wittig-Horner reaction between an anion of the phosphine oxide 18 and the
ketone 19a . Two conformers are shown with a torsional angle P-C(7)-C(8)-O set at 0°; all hydrogen atoms are omitted for
clarity.
Sch em e 2
hindrance with phosphorus substituents and, as a
consequence, do not prevent formation of the desired
diene product 20a .
Con for m a tion a l An a lysis. It has been established
that ring A of vitamin D3, 1R-hydroxyvitamin D3, and
the natural hormone 113 and its analogues14 exist in
solution as two chair forms (named R and â, Figure 4a)
in rapid equilibrium.15 Of particular interest are com-
pounds in which, due to some modifications of their
structures, such conformational equilibrium is strongly
shifted to one particular A-ring chair form. Biological
testing of such compounds could possibly provide some
insight into an intriguing question as to which confor-
mation of ring A in vitamin D compounds is responsible
for its biological action. In an attempt to solve this
problem, the 2-methyl analogues of the natural hor-
Analogous to the procedure described above, Wittig-
Horner coupling of 17 and 18 with the protected (20S)-
25-hydroxy Grundmann’s ketone 19b provided 19-
norvitamin D3 compounds 20b and 21b in 90 and 15%
yields, respectively. Deprotection of silyl protecting
groups in the obtained condensation products 20a ,b and
21a ,b gave 2-ethylidene-19-norvitamins 8a ,b and 9a ,b
(56-75% yield), respectively. They were, in turn, sub-
jected to homogeneous catalytic hydrogenation with
Wilkinson’s catalyst (Scheme 2), and the corresponding
pairs of 2R- and 2â-ethyl-19-norvitamins 6a ,7a and
6b,7b, formed in ca. 45% yield, were easily separated
by HPLC. The stereochemistry at C-2 in the synthesized
vitamin D compounds was tentatively determined on
the basis of conformational analysis, molecular model-
mone16 and 19-nor-1R,25-(OH)2D3 were synthesized.
6
Clearly, the presence of a bulky 2-alkyl substituent
results in a significant bias toward one chair form of
the A-ring (Figure 4b). Therefore, the 2-ethyl group,
characterized by a large conformational free energy A
value (1.75 kcal/mol)17 similar to that of the methyl
1
ing, and H NMR spectroscopy.

3370 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Sicinski et al.
F igu r e 4. Conformational equilibrium in ring A of 1R-hydroxyvitamin D3 analogues (a), monosubstituted cyclohexanes (b), and
2-substituted alkylidenecyclohexanes (c).
group (1.70 kcal/mol),17 should exert a similar effect on
the A-ring conformation, shifting the equilibrium toward
the conformers with the equatorial C-2-ethyl substitu-
ents. In the obtained 2-ethylidene-19-norvitamin D3
compounds, a different strong interaction (designated
as A(1,3)-strain,18,19 Figure 4c) existing between the
methyl group from the ethylidene moiety and equatorial
hydroxyls at C-1 or C-3 should be involved. This should
result in the strong bias toward conformers with an
axial orientation of this hydroxy group to which the
methyl from the ethylidene fragment is directed.
Molecular modeling fully supported these assump-
tions regarding the structure of vitamin D compounds
described in the present work. The preferred A-ring
conformations of isomeric 2-ethyl- and 2-ethylidene-
substituted 19-norvitamins (Figure 5) were established
by force field calculations (PC MODEL 6.0, Serena
Software)20 performed on model compounds lacking side
vitamin 8a , large coupling constants of the same
10R-H, resonating at δ 1.85 (br t, J ∼ 12 Hz), indicated
its trans-diaxial relationship with the vicinal 1â-H and,
consequently, significant bias toward a chair conforma-
tion possessing 1R- and 3â-hydroxyls in an equatorial
and axial orientation, respectively.
As expected, 1H NMR spectra of 2-ethyl vitamins 6a ,b
and 7a ,b were very similar to those of the previously
synthesized 2-methyl analogues 4a ,b and 5a ,b.
Biologica l Eva lu a tion . The problem of the A-ring
form required for biological activity of vitamin D has
been discussed since 1974 when Okamura et al.22
postulated that calcium regulation ability is limited to
vitamin D compounds which can assume a conformation
with the 1R-hydroxy group (commonly considered as the
substituent crucial for biological activity) occupying the
equatorial position. According to this hypothesis, such
geometry of the A-ring is required for binding to the
vitamin D receptor and induction of subsequent biologi-
cal responses involving transport and mobilization of
the calcium in the body. The results of our biological
studies on 1R,25-dihydroxy-10,19-dihydrovitamin D3
analogues described in 199423 stood in some contrast
with Okamura’s suggestion because it was found that
the 1R-hydroxy group in the most calcemic 10S-isomer,
1
chains. Also, analysis of the H NMR spectra of the final
vitamins leads to the same conclusions. Thus, for
example, the magnitudes of vicinal coupling constants
(13.7, 3.9 Hz) of the 10R-H proton signal (δ 2.87) in the
2R-ethyl vitamin 6a support its equatorial disposition,21
whereas the multiplet pattern of 4â-H at δ 2.14 (t, J ∼
11 Hz) is typical of an axial hydrogen. However, in

Analogues of 1R,25-Dihydroxy-19-norvitamin D3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3371
F igu r e 5. Preferred, energy-minimized (PC MODEL 6.0, Serena Software) A-ring conformations of the synthesized analogues
6a ,b (a), 7a ,b (b), 8a ,b (c), and 9a ,b (d). Steric energy differences between the preferred conformers and their partners with the
inverted chair forms (calculated for model compounds lacking side chain) are given. Corresponding percentage populations (in
parentheses) of conformers are given for room temperature (25 °C).
possessing the strongly preferred A-ring conformation,
occupies an axial position. Recent structure-activity
studies performed on 2-substituted analogues of natural
the complexes with the natural ligand and its 20-epi
analogues is the position of the methyl group at C-20
which is situated in the latter compounds closer to His
397. The fact that 20-epi compounds protect the VDR
against degradation27 more efficiently than the natural
hormone 1 and the observation that double mutation
(at positions 421 and 422)28 in hVDR is important only
for binding of 20-epi analogues but not for the hormone
seem to suggest different conformations of VDR in the
complexes mentioned above. These results are in con-
trast with the crystal structures of the hVDR deletion
mutant complexes (118-425, ∆[165-215]) with the
hormone or 20-epi analogues26 where the overall con-
formation of hVDRmt and the position of H12 are
strictly maintained.
It should also be noted that one of the mutations of
hVDR (S278A) was described29 that did not affect the
affinity for 1 and, therefore, did not confirm a contact
between Ser 278 and the ligand’s hydroxyl at C-3. This
may suggest that the in vivo conformation of the
liganded 1 can be different from that reported for its
crystals with hVDRmt. When the facts described above
are considered, it could be concluded that the role of 1R-
hydroxyl orientation and A-ring conformation in the
biological profile of the vitamin D molecule is still
unclear, and additional information is needed.
hormone16 and 19-nor-1R,25-(OH)2D3 indicated a simi-
6
lar relationship between the orientation of 1R-OH24 and
biological activity. Thus, in the preferred A-ring con-
formation of these vitamins, a bulky alkyl (hydroxy-
alkyl) substituent occupies an equatorial orientation.
Consequently, the 1R-hydroxyl in 2R- and 2â-substituted
vitamins must be oriented axially and equatorially,
respectively. Biological data strongly indicate that 2R-
alkyl vitamins have considerably higher calcemic activ-
ity than their 2â-substituted counterparts, and this
trend is particularly remarkable in the 20S-series. An
interesting input to the problem of importance of one
particular A-ring conformation of vitamin D analogues
came from the very recent studies of the crystal struc-
ture of the VDR. Although the VDR and its complex
with hormone 1 resisted all attempts at crystallization,
the Moras group succeeded in getting the crystals of 1
complexed with the hVDR mutant (118-425, ∆[165-
215]).25 The reported X-ray crystallographic data of this
receptor, complexed with 1 as well as with its analogues
with the modified side chain,26 revealed that in all cases
these vitamins were present with their A-rings in
â-chair form and with equatorially oriented 1R-hydroxy
groups. Moreover, the hydroxy groups in the examined
analogues, including 20-epi compounds MC 1288 and
KH 1060, bound the receptor making the same contacts
within the binding pocket. The main difference between
The synthesized vitamins 6a ,b-9a ,b were tested for
their ability to bind the porcine intestinal vitamin D
receptor. The presented results (Table 1) indicate that
2R-ethyl-19-norvitamins 6a and 6b bind the receptor

3372 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
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
5.0 × 10-10
6.0 × 10-10
5.0 × 10-8
2.5 × 10-10
3.0 × 10-9
1
1.2
100
0.5
6.0
2.7 × 10-9
4.6 × 10-10
1.2 × 10-8
1.3 × 10-10
6.3 × 10-9
1
2R-ethyl-19-nor-1R,25-(OH)2D3
2â-ethyl-19-nor-1R,25-(OH)2D3
6a
7a
6b
7b
0.17
4.4
0.05
2.3
2R-ethyl-19-nor-(20S)-1R,25-(OH)2D3
2â-ethyl-19-nor-(20S)-1R,25-(OH)2D3
1R,25-(OH)2D3
1
6.0 × 10-10
2.5 × 10-10
7.0 × 10-9
2.8 × 10-10
5.0 × 10-10
1
0.4
12
0.5
0.8
2.5 × 10-9
2.0 × 10-9
4.0 × 10-9
2.2 × 10-10
1.5 × 10-10
1
2-ethylidene-19-nor-1R,25-(OH)2D3 (E-isomer)
2-ethylidene-19-nor-1R,25-(OH)2D3 (Z-isomer)
2-ethylidene-19-nor-(20S)-1R,25-(OH)2D3 (E-isomer)
2-ethylidene-19-nor-(20S)-1R,25-(OH)2D3 (Z-isomer)
8a
9a
8b
9b
0.8
1.6
0.09
0.06
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. The binding ratio
b
is 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. The 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. The differentiation activity ratio is the
ratio of the analogue average ED50 to the ED50 for 1R,25-(OH)2D3.
better than their isomers with the 2â-ethyl substituent.
In the series of 2-ethyl analogues, 20-epimerization
increases the binding potency, especially (17 times) for
the 2â-isomers (7a versus 7b). Similar trends were
observed previously for 2-methyl-substituted 19-norvi-
tamins6 and derivatives of the natural hormone.16d,e The
competitive binding analysis showed that 2-ethylidene-
19-norvitamins possessing the methyl group from the
ethylidene moiety directed toward C-3, that is, trans in
relation to the C(6)-C(7) bond (E-isomers), are more
active than 1R,25-(OH)2D3 in binding to VDR; their
counterparts with the cis relationship between the
ethylidene methyl substituent and C(7)-H group (Z-
isomers) exhibit reduced affinity for the receptor. It is
interesting that 20-epimerization does not influence the
binding ability of the E-isomer (8a versus 8b), while it
significantly increases (15 times) the potency of the
Z-isomer form (9a versus 9b). Our previous work
indicated that the binding potency of 2-methylene-
substituted 19-norvitamins is not affected by 20-epimer-
ization.6
of the 1R-hydroxy group in their preferred A-ring
conformations.
Receptor Modelin g an d Ligan d Dockin g. 1. Model
of th e F u ll-Len gth Liga n d Bin d in g Dom a in of Ra t
VDR. The vitamin D receptor (VDR) shares its “R-
helical sandwich structure” with other members of the
nuclear receptor (NR) superfamily which includes re-
ceptors for the steroid, retinoid, and thyroid hor-
mones.29-31 Interestingly, ligands which bind to the
respective proteins (ER, PR, RAR, and VDR) are har-
bored in the same region in all members of the NR
family.29 The VDR is most closely related in architecture
to the RAR. Such structural similarity, delineated from
homology modeling, was verified by the crystal structure
of the hVDR (118-425, ∆[165-215]) mutant.25
Although the architecture of the vitamin D receptor
resembles those of other nuclear receptors, it contains
one distinct feature: a long insertion domain connecting
helices H1 and H3. This highly nonhomologous frag-
ment covers 72-81 residues in the VDR family com-
pared to 15-25 residues in other members of the NR
family. The domain, encompassing residues 165-215 in
hVDR, is poorly conserved in different VDR species (9%
identity between amino acids 157 and 215). Until now,
only one biological function (phosphorylation) has been
found for this unique fragment.32 Its presence hampers
the efforts directed toward establishing the VDR struc-
ture because this fragment is probably responsible for
the decreased crystallization ability of the protein and,
at the same time, creates serious problems in protein
homology modeling. Therefore, it is not surprising that
even in a recently calculated hVDR molecular model,33
fragment 162-221 was excised because of the absence
of any structural information on which homology model-
ing could be based. However, we have reported that by
employing lattice modeling (SICHO (SIde-CHain Only)
method) for a folding of poorly structured parts (residues
120-164) of the rat VDR, a structure highly consistent
with the crystal structure of the hVDR mutant was
retrieved.34
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. Both 2R-ethyl-19-norvita-
mins and both isomers (E and Z) of (20S)-2-ethylidene-
19-norvitamin D3 are more potent than 1R,25-(OH)2D3
in this assay (Table 1), whereas the remaining tested
compounds are almost equivalent to hormone 1. Both
E-isomers of the 2-ethylidene-19-norvitamins, when
tested in vivo in rats (Table 2), exhibited high calcemic
activity with the 20S-compound being especially potent.
The isomeric Z compounds are significantly less potent,
having no intestinal calcium transport activity. 2-Ethyl-
19-norvitamins have some ability to mobilize calcium
from bone but not to the extent of hormone 1; however,
they are inactive in the intestine. The only exception is
the 2R-ethyl isomer from the 20S-series which exhibits
a strong calcium mobilization response and marked
intestinal activity. Comparison of biological in vitro and
in vivo activities of 2-ethyl- and 2-ethylidene-19-norvi-
tamins exhibits, rather to our surprise, that their
potency seems to not be influenced by the orientation
In this paper, the first model of the full-length ligand
binding domain of rVDR (118-423) was constructed.
The same method, which combined homology and lattice

Analogues of 1R,25-Dihydroxy-19-norvitamin D3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3373
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
amount
(pmol)
Ca transport S/M
(mean ( SEM)
serum Ca
(mean ( SEM)
compound
compd no.
none (control)
1R,25-(OH)2D3
2R-ethyl-19-nor-1R,25-(OH)2D3
2â-ethyl-19-nor-1R,25-(OH)2D3
2R-ethyl-19-nor-(20S)-1R,25-(OH)2D3
2â-ethyl-19-nor-(20S)-1R,25-(OH)2D3
0
260
260
260
260
260
3.8 ( 0.4b
6.5 ( 0.9c
4.0 ( 0.4d
3.7 ( 0.3e
5.0 ( 0.4f
4.1 ( 0.3g
3.9 ( 0.1b
5.8 ( 0.1c
5.1 ( 0.1d
5.0 ( 0.1e
7.0 ( 0.1f
5.6 ( 0.1g
1
6a
7a
6b
7b
none (control)
1R,25-(OH)2D3
0
130
260
65
130
65
3.0 ( 0.7b
4.3 ( 0.1b
1
1
1
5.5 ( 0.5c
5.1 ( 0.3c
2
5.9 ( 0.4c2
5.8 ( 0.3c
1
2-ethylidene-19-nor-1R,25-(OH)2D3 (E-isomer)
2-ethylidene-19-nor-1R,25-(OH)2D3 (Z-isomer)
8a
9a
5.0 ( 0.4d
4.5 ( 0.1d1
5.2 ( 0.2d2
4.4 ( 0.2e1
4.2 ( 0.0e2
6.8 ( 0.4d2
4.4 ( 0.4e1
5.7 ( 0.9e2
130
none (control)
1R,25-(OH)2D3
0
130
260
65
4.4 ( 0.2b
4.9 ( 0.7c1
6.0 ( 0.9c2
9.0 ( 0.3d1
4.1 ( 0.2b
5.2 ( 0.2c1
1
2
6.4 ( 0.4c
1
2-ethylidene-19-nor-(20S)-1R,25-(OH)2D3 (E-isomer)
8b
8.2 ( 0.3d
2
2
130
5.8 ( 0.8d
12.1 ( 0.6d
none (control)
1R,25-(OH)2D3
0
65
130
65
4.6 ( 0.4b
5.2 ( 0.6c1
6.0 ( 0.7c2
4.3 ( 0.1b
1
1
5.7 ( 0.1c
2
6.4 ( 0.1c
1
1
2-ethylidene-19-nor-1R,25-(OH)2D3 (Z-isomer)
9b
6.6 ( 0.8d
4.4 ( 0.1d
2
2
130
6.7 ( 0.2d
5.1 ( 0.3d
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. Over 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 per group. Statistical analysis was done by Student’s t test. Statistical
data: serosal/mucosal (S/M), panel 1, b from c, p < 0.01, b from d, e, and g, NS, b from f, p ) 0.01; panel 2, b from c1, d1, and e1, NS, b
from c2, p ) 0.001, b from d2, p < 0.001, b from e2, p < 0.025; panel 3, b from c1 and d2, NS, b from c2, p ) 0.025, b from d1, p < 0.001;
panel 4, b from c1, NS, b from c2, d1, and d2, p < 0.005; serum calcium, panel 1, b from c, f, and g, p < 0.001, b from d and e, p < 0.005;
panel 2, b from c1 and d2, p < 0.025, b from d1, e1, and e2, NS, b from c2, p < 0.001; panel 3, b from c1, p < 0.005, b from c2, d1, and d2,
p < 0.001; panel 4, b from c1 and c2, p ) 0.001, b from d1 and d2, NS.
is also known that removal of the domain 165-215 has
no major effect on ligand binding, transactivation, or
dimerization of the vitamin D receptor with RXR.25 Our
model shows that the region connecting helices H1 and
H3 is distant from the pocket and, therefore, is unlikely
to affect the binding abilities of the ligand. This struc-
ture is confirmed by an experiment indicating that
mutation C190W (hVDR) does not influence the binding
function of the receptor.37a Moreover, according to our
computational model, the disordered domain (164-207)
is sufficiently remote from the receptor region involved
in transcription (H12) and dimerization with RXR (H3).
Further evidence supporting our model comes from the
observation that hVDR is sensitive to proteolysis with
a trypsin cleavage site after Arg 174.37b The region in
F igu r e 6. Stereoview of the three-dimensional structure of
rat VDR. The nonhomologous part of the receptor between long
rVDR, compatible with the above-mentioned domain,
helices H1 and H3 (residues 164-207, colored in blue) was
begins with Thr 171 which is situated on the remote
modeled by SICHO.
edge of the protein (Figure 6).
Superimposition of the full-length rat VDR-LBD
modeling and was used successfully for creating two
simplified rVDR models,34 was also employed in this
work. The nonhomologous portion of the VDR receptor,
excised in crystallized hVDRmt, was modeled by the
program SICHO35 and is shown in Figure 6. It is readily
observed that the lattice-modeled domain (residues
164-207) is highly disordered and does not contain any
well-structured elements. This unique VDR part of the
protein forms loops, spanning between amino acids Ser
178-Asn 204 and Ser 205-Ser 218. The poorly struc-
tured architecture of the domain, delineated from lattice
modeling, remains in excellent agreement with struc-
tural investigations (SAXS) performed in solution.36 It
model and RAR crystal structure is shown in Figure 7.
With the exclusion of the highly disordered VDR loop
(164-207), the proteins overlaid with an RMSD of 2.52
Å over 156 residues (compared by using the Gerstein
and Levitt structural alignment algorithm).38 It is worth
mentioning that experimental data supporting our full
model of rVDR come from different kinds of experi-
ments, covering structural investigation in solution as
well as biological activities.
2. Dock in g Exp er im en ts. Until the present time,
only complexes of the hVDR deletion mutant (118-425,
∆ [165-215]) with vitamin D analogues possessing an

3374 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Sicinski et al.
F igu r e 7. Stereoview of the superimposition of the rat VDR-
LBD model (blue) and human RAR (red). Part of rVDR
modeled by SICHO (residues 164-207) is colored in light blue.
unmodified A-ring (i.e., analogous to that of the hor-
mone) have been crystallized. It was found that side
chain-modified analogues with a natural configuration
at C-20 (MC 903, EB 1089, and TX 522) bind to the
hVDRmt in a fashion similar to that of 1R,25-(OH)2D3.26a
Thus, the A-ring is always oriented toward Tyr 143, and
the side chain hydroxyl at C-25 contacts the same amino
acids (His 305 and His 397). Also, the 1R-OH group in
the analogues creates hydrogen bonds with Ser 237 and
Arg 274, while 3â-OH creates bonds with Ser 278 and
Tyr 143.25
In the present work, we performed docking simula-
tions of the four synthesized vitamins 6a -9a possessing
the same side chain as the natural hormone 1. When
the significant differences in their binding affinity for
VDR were taken into account, it was interesting to
establish how they could accommodate the binding
pocket. These four vitamins were docked into full-length
rVDR-LBD in their energy-minimized conformations
(see Experimental Section). The docking procedure
employed in this work was verified by modeling com-
plexes between full-length rVDR-LBD and natural
hormone 1 that was introduced into the binding pocket
by an A-ring or side chain. Regardless of such different
positioning, 1R,25-(OH)2D3 was finally oriented in the
pocket in the “native” fashion. Our docking experiments
performed with 19-norvitamins 6a -9a revealed that the
binding pocket consists of eleven amino acids common
for all modeled complexes: His 225, Leu 226, Val 230,
Ile 264, Glu 265, Met 268, Trp 282, Cys 284, Lys 290,
Tyr 291, and His 393 (Figure 8, residues marked in
green). It is worth mentioning that His 393 also forms
hydrogen bonds to 25-OH in the “native” complex.
Involvement of the amino acids His 225, Val 230, Trp
282, Cys 284, and His 393 in hormone binding was
verified by mutations34 as well as affinity labeling39 and
is in agreement with contacts found in the rVDR pocket
that accommodated 19-nor analogues. The existence of
contacts between glutamic acid (Glu 265) and A-ring-
functionalized analogues is supported by newly pub-
lished data.27 The studies on point mutation E269A of
hVDR allowed the authors to suggest that Glu 269 may
have a role in stabilizing the conformation of the
receptor after ligand binding. The presence of Trp 282
and Cys 284 in the VDR binding pocket was confirmed
by several methods.25,40 It is worth noting that in all
F igu r e 8. View of the three-dimensional structure of the
ligand binding cavity of the modeled rat VDR with the docked
vitamin D analogue 8a . The common contacts (distances
shorter than 3.5 Å) found between any atom of the ligands
(6a -9a ) and receptor are marked in green. (a) Residues of the
human VDRmt (118-425, ∆[165-215]), creating contacts with
the hydroxy groups of the hormone, are marked in orange. His
393, being in contact sites for the hormone and the 19-
norvitamins 6a -9a , is marked in red. Ring A of the hormone
is directed toward Tyr 143, whereas for the analogues 6a -
9a , it is oriented between Tyr 143 and Phe 150. Tryptophan
is situated close to the intercyclic diene fragment of the
hormone and 19-norvitamins, and its indole ring is directed
toward the A-ring of the ligands. (b) The most important
hydrogen bonds with ligand’s hydroxyls and Cys 284 and Met
268 are marked in magenta. Spatial regions within 3.5 Å of
the contact residues lining the pocket are shown as spheres
(green dots).
modeled complexes of analogues 6a -9a with the full-
length rVDR-LBD, tryptophan is located under the
vitamin 5,7-diene fragment (similarly as in the crystal-
line “native” complex)25 and directs its indole-ring
toward the A-ring of the vitamin D compound (Figure
8a). Interestingly, the same set of amino acids creates
hydrogen bonds with the hydroxyls of 2-ethyl and
2-ethylidene analogues. It was found that Met 268 and
Tyr 291 contact 25- and 3â-hydroxy groups, respectively.
Cys 284 plays an important role in the binding of
2-ethylidene-19-norvitamins. In both complexes of ana-
logues 8a and 9a , this amino acid bridges their A-ring
hydroxyls; the formation of hydrogen bonds to 1R- and
3â-OH groups can be especially effective in the case of
the former E-isomer (Figure 8b) which, interestingly,
shows the best ability to bind the porcine intestinal
VDR. Also in the complex of 2â-ethyl compound 7a , Cys

Analogues of 1R,25-Dihydroxy-19-norvitamin D3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3375
284 contacts its 1R-OH substituent. In the case of the
isomeric 2R-ethyl analogue 6a , its hydroxyl at C-1 is
hydrogen-bonded to His 225 and Tyr 291. All 2-ethyl-
and 2-ethylidene-19-norvitamins dock into the VDR
binding pocket with their side chains in the lowest-
energy (extended) conformation that stays in contrast
with a side chain orientation detected for the hormone
1 in its crystalline complex.25
effect on biological potency. Also, the comparison of
biological activities of the most active analogues in the
2-alkyl and 2-ethylidene series studied by us so far, that
is, possessing 2R-methyl (ethyl) and (E)-2-ethylidene
substituents, indicates that their potency is not deter-
mined only by the axial and equatorial orientation,
respectively, of the 1R-hydroxy group in their preferred
A-ring conformations. It is likely that differences in the
accommodation of 2-alkyl (alkylidene) groups in the
VDR pocket and their interactions with the surrounding
amino acids can play a more important role. Biological
data of 2-methylene-6 and 2-ethylidene-19-norvitamin
D3 analogues show that such homologation of the
alkylidene group at C-2 in the compound from the 20S-
series provides the analogue (E-isomer) that has strong
activity on bone and, additionally, is very effective on
intestinal calcium transport.
Generally, compounds with an “unnatural” configu-
ration at C-20 are characterized by significantly in-
creased potency; explanation of this phenomenon would
require additional studies. Over the last two years,
several articles appeared highlighting an influence of
20-epimerization on biological activities of vitamin D
compounds.26a,27,42,43 It was found that a greater tran-
scriptional potency (100 times) of 20-epi-1R,25-(OH)2D3
is attributed primarily to its ability to enhance dimer-
ization of VDR.42 Recent studies also demonstrate the
differences in target tissue metabolic paths of 1R,25-
dihydroxy-20-epi vitamin D3 and suggested that they
are responsible for the increased biological activities of
20-epi vitamin D3 analogues.43
The consistency of our full-length rVDR-LBD compu-
tational model with the experimentally outlined struc-
ture proves that lattice modeling of poorly homologous
domains results in a model resembling the structure of
the native protein. Automatic docking simulations
performed by Flexi Dock software correctly positioned
ligands in the VDR pocket, even when they were
initially inversely situated in the protein cavity. Various
analogues with different orientations of the 1R-OH
group (axial or equatorial) docked in the 6-(s)-trans
conformation bind VDR in a similar way, being situated
in the binding pocket with their A-ring oriented toward
the interior of the cavity. 19-Norvitamin D3 compounds
modified with nonpolar C-2-substituents could be easily
positioned in the receptor binding pocket because the
amino acids lining the rVDR domain, the closest to C-2,
are mostly hydrophobic. Although the differences in
energy between complexes are small, they correspond
to the binding potency of the analogues.
It was established that differences in the energies of
the studied VDR complexes of 19-norvitamins 6a -9a
do not exceed 30 kcal/mol. Interestingly, analogues 7a
and 8a , characterized by the greatest difference in
binding affinity (100 times lower and 2.5 times higher
than 1, respectively), create complexes with significantly
different energies of -85 and -102 kcal/mol (Figure 8),
respectively, corresponding to the binding ability of the
compounds. It seems that the highest binding potency
of compound 8a results not only from the strong contacts
between its three hydroxyls and the receptor but also
from stabilization of the ethylidene fragment by the
amino acids Y 147, F 150, H 225, C 284, Y 289, and Y
291 which form channels surrounding this substituent.
An aromatic ring of Phe 150 that is positioned parallel
(3 Å) to the double bond of the ethylidene group can
effectively stabilize it via π-π interactions. Of the two
2-ethyl-19-norvitamin D3 analogues 6a and 7a , the
former compound with the alkyl group at C-2 in the
R-orientation (“up”) binds VDR more effectively (83
times) than the latter (“down”) 2â-isomer. A similar
trend was observed for the C-2-alkylated hormone and
its 5E-analogues.16d,e,41 Inspection of our ligand-rVDR
models of 6a and 7a indicates that the receptor creates
weaker contacts to three hydroxy groups of the former
compound. It seems that hydrophobic Leu 229, which
is present in the vicinity of the 2R-ethyl group in the
complex of 6a , stabilizes this substituent very ef-
fectively, and it could be responsible for the improve-
ment of the binding ability of the analogue. We estab-
lished previously6 that introduction of the 2R-methyl
group into the 1R,25-dihydroxy-19-norvitamin D3 mol-
ecule slightly (1.4 times) decreases its binding potency
to VDR. In contrast, introduction of the 2R-ethyl sub-
stituent results in increased VDR binding affinity (2.8
times) of the analogue. Interestingly, a completely
opposite trend was observed for the C-2-functionalized
hormone, where 2R-methyl substitution increased (4
times) the binding potency while insertion of 2R-ethyl
decreased (2.5 times) it.41 This might indicate that
contacts in the binding pocket of A-ring-substituted
analogues of the vitamin hormone differ from those of
their 19-nor counterparts. Such a conclusion, deducted
from binding activity studies, is in agreement with our
docking computer experiment.
Exp er im en ta l Section
Ch em istr y. Optical rotations were measured in chloroform
using a Perkin-Elmer 241 automatic polarimeter at 22 °C.
Ultraviolet (UV) absorption spectra were recorded with a
Hitachi model 60-100 UV-vis spectrometer in the solvent
noted. 1H and 13C nuclear magnetic resonance (NMR) spectra
were recorded at 500 and 125 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
Con clu sion s
The presented results confirm our earlier observations
indicating that the introduction of different C-2-sub-
stituents into the parent 19-nor-1R,25-dihydroxyvitamin
D3 molecule can drastically change the biological activity
of the analogues. Comparison of the biological potency
of the synthesized 2R-ethyl analogues with the previ-
ously reported data6 for the corresponding 2R-methyl-
19-norvitamins clearly indicates that the increased size
of the hydrophobic alkyl group at C-2 has an adverse

3376 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
model 6000A solvent delivery system, a model 6 UK
Universal injector, a model 486 tunable absorbance detector,
and a differential R 401 refractometer. Microanalysis of
crystalline compounds was within (0.4% of the theoretical
values. THF was freshly distilled from sodium benzophenone
ketyl under argon before use.
Sicinski et al.
a
washed with brine, dried (MgSO4), and evaporated. The
residue was dissolved in hexane, applied on a silica Sep-Pak
cartridge, and washed with hexane (20 mL) to remove apolar
impurities. Elution with hexane/ethyl acetate (99.2:0.8, 20 mL)
gave impure 4′-ethylidene compounds 13 and 14 as a colorless
oil (ca. 8 mg). Purification of the products was achieved by
HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/min) using
the hexane/ethyl acetate (98:2) solvent system. Pure com-
pounds 13 and 14 were eluted at RV 34 mL (single peak) as a
colorless oil (4.4 mg, 18%; 13:14 isomer ratio of 1:1.7).
The starting hydroxy ketone 10 was obtained from com-
mercial (-)-quinic acid as described previously.10 10: mp 98-
1
100 °C (from hexane); [R]22 -19.2° (c 0.9, CHCl3); H NMR
D
(CDCl3) δ 0.067, 0.086, and 0.092 (6H, 3H, and 3H, each s, 4
× SiCH3), 0.856 and 0.899 (9H and 9H, each s, 2 × Si-t-Bu),
2.25 (1H, ddd, J ) 14.4, 4.0, 1.8 Hz), 2.45 (1H, ddd, J ) 14.0,
5.2, 1.0 Hz), 2.60 (1H, dd, J ) 14.0, 10.2 Hz), 2.77 (1H, ddd, J
2-[(3′R,5′R)-3′,5′-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-4′-
eth ylid en ecycloh exylid en e]eth a n ol (15 a n d 16). Diisobu-
tylaluminum hydride (1.5 M in toluene, 0.2 mL, 0.3 mmol) was
slowly added to a stirred solution of the allylic esters 13 and
14 (1:1.7, 29.6 mg, 67 µmol) in toluene/methylene chloride (2:
1, 1.2 mL) at -78 °C under argon. The mixture was stirred at
-78 °C for 1 h; the reaction was quenched by the addition of
potassium sodium tartrate (2 N, 1 mL), aq HCl (2 N, 1 mL),
and H2O (12 mL), and then the mixture was diluted with ether
and benzene. The organic layer was washed with diluted
NaHCO3 and brine, dried (MgSO4), and evaporated. The
residue was purified by flash chromatography. Elution with
hexane/ethyl acetate (95:5) gave semicrystalline allylic alcohols
15 and 16 (22.1 mg, 80%, 15:16 isomer ratio of 1:1.8).
Separation of both isomers was achieved by HPLC (10 mm ×
25 cm Zorbax-Sil column, 4 mL/min) using the hexane/ethyl
acetate (9:1) solvent system. Pure compounds 15 (oily) and 16
(crystalline) were eluted at RV 44 and 47 mL, respectively.
) 14.4, 3.5, 1.0 Hz), 3.80 (1H, ∼d, J ) 4 Hz), 4.27 (2H, m); 13
C
NMR (CDCl3) δ -5.08 (q), -4.93 (q), -4.88 (q), -4.74 (q), 17.86
(s), 18.02 (s), 25.59 (q), 25.74 (q), 44.28 (t), 45.98 (t), 69.41 (d),
70.04 (d), 72.56 (d), 207.32 (s).
(3R,5R)-3,5-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-1,4-cyclo-
h exa n ed ion e (11). To a stirred mixture of ruthenium(III)
chloride hydrate (331 mg, 1.6 mmol) and sodium periodate
(8.37 g, 39.2 mmol) in water (32 mL) was added a solution of
hydroxy ketone 10 (4.05 g, 10.8 mmol) in CCl4/CH3CN (1:1,
48 mL). The mixture was vigorously stirred at room temper-
ature for 22 h. A few drops of 2-propanol were added, and the
mixture was poured into water and extracted with toluene.
The organic layer was washed with 1% NaHSO3 and brine,
dried (MgSO4), and evaporated to give a dark oily residue
which was purified by flash chromatography. Elution with
hexane/ethyl acetate (9:1) gave pure, semicrystalline diketone
11 (2.04 g, 65% based on recovered hydroxy ketone 10) and
unchanged substrate (0.88 g). 11: [R]22D -40.2° (c 0.5, CHCl3);
1H NMR (CDCl3) δ 0.073 and 0.094 (each 6H, each s, 4 ×
SiCH3), 0.888 (18H, s, 2 × Si-t-Bu), 2.71 (2H, ddd, J ) 15.0,
7.4, 1.9 Hz), 2.89 (2H, ddd, J ) 15.0, 5.2, 1.9 Hz), 4.66 (2H,
dd, J ) 7.4, 5.2 Hz); 13C NMR (CDCl3) δ -5.22 (q), -4.94 (q),
-4.81 (q), -4.74 (q), 18.01 (s), 18.15 (s), 25.59 (q), 25.69 (q),
46.89 (t), 49.55 (t), 71.79 (d), 72.05 (d), 203.18 (s), 206.12 (s);
MS m/z (relative intensity) no M+ 357 (M+ - Me, 4), 315 (M+
- t-Bu, 100), 297 (3), 271 (14), 183 (32), 73 (85).
[(3′R,5′R)-3′,5′-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-4′-oxo-
cycloh exylid en e]a cetic Acid Meth yl Ester (12). To a
solution of diisopropylamine (0.44 mL, 3.14 mmol) in anhy-
drous THF (2.2 mL) was added n-BuLi (2.5 M in hexanes, 1.26
mL, 3.15 mmol) under argon at -78 °C; the mixture was
stirred, and methyl(trimethylsilyl)acetate (0.52 mL, 3.17
mmol) was then added. After 15 min, the diketone 11 (819.8
mg, 2.2 mmol) in anhydrous THF (2.2 + 1 mL) was added
dropwise over 8 min. The solution was stirred at -78 °C for
an additional 12 min, and the reaction was quenched with wet
ether. The mixture was poured into brine and extracted with
ether and benzene. The combined extracts were washed with
brine, dried (MgSO4), and evaporated. The light-brown oily
residue was purified by flash chromatography. Elution with
hexane/ethyl acetate (95:5) gave a pure allylic ester 12 (601.8
mg, 64%) as a colorless oil which slowly crystallized upon
standing in the refrigerator.
[(3′R,5′R)-3′,5′-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-4′-eth -
ylid en ecycloh exylid en e]a cetic Acid Meth yl Ester s (13
a n d 14). To the ethyltriphenylphosphonium bromide (104 mg,
0.28 mmol) in anhydrous THF (1.3 mL) at 0 °C was added
dropwise n-BuLi (1.6 M in hexanes, 0.18 mL, 0.29 mmol) under
argon while the mixture was stirred; the solution was stirred
at 0 °C for 10 min, and then at room temperature for 10 min.
The orange-red mixture was cooled to -78 °C and siphoned
in 3 equal portions (20 and 50 min intervals between the next
portions) into a solution of ketoester 12 (24 mg, 56 µmol) in
anhydrous THF (0.5 mL) at -78 °C. The reaction mixture
(yellow solution with colorless precipitate) was stirred at -78
°C, and the reaction was quenched by the addition of brine
containing 1% HCl (3 h 40 min after addition of the first
portion of the Wittig reagent), AcOEt (15 mL), benzene (10
mL), ether (5 mL), and saturated NaHCO3 (10 mL) were
added, and the mixture was vigorously stirred at room
temperature for 18 h. The organic phase was separated,
[2-[(3′R,5′R)-3′,5′-Bis[(ter t-b u t yld im et h ylsilyl)oxy]-4′-
eth yliden ecycloh exyliden e]eth yl]diph en ylph osph in e Ox-
id es (17 a n d 18). (1) To the allylic alcohols 15 and 16 (1:1.7,
21.7 mg, 53 µmol) in anhydrous THF (0.5 mL) was added
n-BuLi (2.5 M in hexanes, 21 µL, 53 µmol) under argon at 0
°C with stirring. Freshly recrystallized tosyl chloride (10.1 mg,
53 µmol) was dissolved in anhydrous THF (100 µL) and added
to the allylic alcohol-BuLi solution. The mixture was stirred
at 0 °C for 5 min and set aside. In another dry flask with air
replaced by argon, n-BuLi (2.5 M in hexanes, 42 µL, 105 µmol)
was added to Ph2PH (18 µL, 100 µmol) in anhydrous THF (150
µL) at 0 °C while the mixture was stirred. 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 ad-
ditional 30 min at 0 °C, and the reaction was quenched by the
addition of H2O (10 µL). Solvents were evaporated under
reduced pressure, and the residue was dissolved in methylene
chloride (0.5 mL) and stirred with 10% H2O2 (0.25 mL) at 0
°C for 1 h. The organic layer was separated, washed with cold
aq sodium sulfite and H2O, dried (MgSO4), and evaporated.
The residue was subjected to flash chromatography. Elution
with hexane/ethyl acetate (9:1) gave unchanged allylic alcohols
(4.0 mg). Subsequent elution with benzene/ethyl acetate (7:3)
provided a mixture of phosphine oxides 17 and 18 (14.7 mg,
46%, 56% based on recovered starting material). (2) Pure
phosphine oxides 17 (60% yield) and 18 (56% yield) were
prepared in the same way from the corresponding individual
allylic alcohols 15 and 16, respectively. Analytical samples of
both isomers were obtained after HPLC (10 mm × 25 cm
Zorbax-Sil column, 4 mL/min) using the hexane/2-propanol (9:
1) solvent system. Pure compounds 17 (oily) and 18 (crystal-
line) were eluted at RV 37 and 39 mL, respectively.
1r-[(ter t-Bu tyld im eth ylsilyl)oxy]-2-eth ylid en e-25-[(tr i-
eth ylsilyl)oxy]-19-n or vita m in D3 ter t-Bu tyld im eth ylsilyl
Eth er (20a a n d 21a ). (1) To a solution of phosphine oxides
17 and 18 (1:1.7, 4.2 mg, 7.0 µmol) in anhydrous THF (140
µL) at -78 °C was slowly added phenyllithium (1.8 M in
cyclohexane/ether, 4 µL, 7.2 µmol) under argon while the
mixture was stirred. The solution turned deep orange. The
mixture was stirred at -78 °C for 20 min, and a precooled
(-78 °C) solution of protected hydroxy ketone 19a (4.5 mg,
11.4 µmol; prepared according to a published procedure9) in
anhydrous THF (100 + 50 µL) was slowly added. The mixture
was stirred under argon at -78 °C for 3 h and at 0 °C for 19
h. Ethyl acetate and water were added, and the organic phase

Analogues of 1R,25-Dihydroxy-19-norvitamin D3
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16 3377
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 (99.7:0.3, 20
mL) to give 19-norvitamin derivatives 20a and 21a (1.6 mg,
29%, 55% based on recovered substrates). The Sep-Pak was
then washed with hexane/ethyl acetate (96:4, 10 mL) to recover
some unchanged C,D-ring ketone 19a (3 mg) and with ethyl
acetate (10 mL) to recover diphenylphosphine oxide (2 mg).
The mixture of protected vitamins was separated by HPLC
(6.2 mm × 25 cm Zorbax-Sil column, 4 mL/min) using the
hexane/ethyl acetate (99.95:0.05) solvent system. Pure com-
pounds 20a (1.4 mg) and 21a (0.2 mg) were eluted at RV 29
and 36 mL, respectively, as colorless oils.
in hexane/ethyl acetate (6:4, 2 mL) and applied on Waters
silica Sep-Pak. A mixture of 2-ethyl vitamins was eluted with
the same solvent system (20 mL). The compounds were further
purified by HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/
min) using the hexane/2-propanol (85:15) solvent system. The
mixture of 2-ethyl-19-norvitamins 6a and 7a (0.52 mg, 47%)
gave a single peak at RV 28 mL, and unreacted substrate (20
µg) was eluted at RV 35 mL. Separation of both epimeric
products was achieved by reversed-phase HPLC (6.2 mm ×
25 cm Zorbax-ODS column, 2 mL/min) using the methanol/
water (8:2) solvent system. 2â-Ethyl vitamin 7a (124 µg, 11%)
was collected at RV 37 mL and its 2R-epimer 6a (362 µg, 33%)
at RV 42 mL.
(2) When pure phosphine oxides 17 and 18 were used as
substrates for Wittig-Horner coupling with 19a (performed
in a manner analogously to the procedure described above),
the corresponding protected 19-norvitamin D3 compounds 20a
and 21a were obtained in 9 and 13% yield, respectively.
(20S)-1r,25-Dih yd r oxy-2r- a n d (20S)-1r,25-Dih yd r oxy-
2â-eth yl-19-n or vita m in D3 (6b a n d 7b). Vitamins 6b and
7b were obtained by hydrogenation of 8b and 9b, performed
in a manner analogous to the process described above for 20R-
isomers 8a and 9a . The hydrogenated compounds were first
purified by HPLC (10 mm × 25 cm Zorbax-Sil column, 4 mL/
min) using the hexane/2-propanol (85:15) solvent system. The
mixture of 2-ethyl-19-norvitamins 7b and 6b (ca. 45%) gave
partially overlapped peaks at RV 27 and 29 mL, respectively.
Separation of both epimers was easily achieved by reversed-
phase HPLC (6.2 mm × 25 cm Zorbax-ODS column, 2 mL/
min) using the methanol/water (8:2) solvent system. 2â-Ethyl
vitamin 7b was collected at RV 30 mL and its 2R-epimer 6b
at RV 45 mL.
(20S)-1r-[(ter t-Bu t yld im et h ylsilyl)oxy]-2-et h ylid en e-
25-[(tr ieth ylsilyl)oxy]-19-n or vita m in D3 ter t-Bu tyld im -
eth ylsilyl Eth er s (20b a n d 21b). Protected 19-norvitamin
D3 compounds 20b and 21b were obtained by the Wittig-
Horner reaction of the protected Grundmann’s ketone 19b
(prepared according to a published procedure6) and the cor-
responding phosphine oxides 17 and 18, performed as de-
scribed for preparation of 20a and 21a . Separation of the crude
products 20b and 21b was achieved by Sep-Pak filtration, and
their final purification was performed by HPLC (10 mm × 25
cm Zorbax-Sil column, 4 mL/min) using the hexane/ethyl
acetate (99.75:0.25) solvent system. Pure compounds 20b and
21b were eluted at RV 25 and 27 mL, respectively, as colorless
oils (90 and 15% yield, respectively).
1r,25-Dih yd r oxy-2-et h ylid en e-19-n or vit a m in D3 (8a
a n d 9a ). The protected vitamin 20a or 21a (2.4 mg, 3 µmol)
in anhydrous THF (200 µL) was treated with tetrabutylam-
monium fluoride (1.0 M in THF, 60 µL, 60 µmol). The mixture
was stirred under argon at room temperature for 23 h, poured
into brine, and extracted with ethyl acetate and ether. Organic
extracts were washed with brine, dried (MgSO4), and evapo-
rated. The residue was purified by HPLC (6.2 mm × 25 cm
Zorbax-Sil column, 4 mL/min) using the hexane/2-propanol (9:
1) solvent system. Analytically pure 2-ethylidene-19-norvita-
min 8a (0.86 mg, 64%) was collected at RV 25 mL, whereas
the isomeric compound 9a (0.75 mg, 56%) was eluted at RV 24
mL (1R,25-dihydroxyvitamin D3 was eluted at RV 49 mL in
the same system) as a white solid. Purity of both compounds
8a and 9a was confirmed by reversed-phase HPLC (6.2 mm
× 25 cm Zorbax-ODS column, 2 mL/min) using the methanol/
water (8:2) solvent system; they were eluted as single sharp
peaks at RV 33 and 40 mL, respectively.
(20S)-1r,25-Dih yd r oxy-2-eth ylid en e-19-n or vita m in D3
(8b a n d 9b). Vitamins 8b and 9b were prepared from the
corresponding protected compounds 20b and 21b by treatment
with tetrabutylammonium fluoride, performed as described for
isomers 8a and 9a from the 20R-series. The products were
purified by HPLC (6.2 mm × 25 cm Zorbax-Sil column, 4 mL/
min) using the hexane/2-propanol (9:1) solvent system. Ana-
lytically pure 2-ethylidene-19-norvitamins 8b (75% yield) and
9b (61% yield) were collected at RV ca. 23 mL. Purity of both
compounds 8b and 9b was confirmed by reversed-phase HPLC
(6.2 mm × 25 cm Zorbax-ODS column, 2 mL/min) using the
methanol/water (8:2) solvent system; they were eluted as single
sharp peaks at RV 25 and 44 mL, respectively.
1r,25-Dih yd r oxy-2r- a n d 1r,25-Dih yd r oxy-2â-eth yl-19-
n or vita m in D3 (6a a n d 7a ). Tris(triphenylphosphine)rhod-
ium(I) chloride (4.6 mg, 5 µmol) was added to dry benzene (2.5
mL) presaturated with hydrogen. The mixture was stirred at
room temperature until a homogeneous solution was formed
(ca. 40 min). A solution of vitamin 8a (1.1 mg, 2.6 µmol) in
dry benzene (0.5 mL) was then added, and the reaction was
allowed to proceed under a continuous stream of hydrogen for
3.5 h. Another portion of catalyst (3.7 mg, 4 µmol) was added,
and the hydrogenation continued for the next 6 h. Benzene
was removed under vacuum, and the residue was redissolved
Biologica l Stu d ies. 1. Mea su r em en t of In testin a l Ca l-
ciu 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. (Indianapolis, IN) and
fed the vitamin D-deficient diet44 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 had no detectable levels of 25-OH-D3 or 1R,-
25-(OH)2D3 in their plasma as measured by methods described
previously.45 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 experi-
ment, the rats were given the indicated doses of compounds
in 0.1 mL of 1,2-propanediol/ethanol (95:5) 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 determine calcium transport
activity and serum calcium concentration. Calcium was mea-
sured in the presence of 0.1% lanthanium chloride by means
of a Perkin-Elmer atomic absorption spectrometer model 3110.
Intestinal calcium transport was determined by the everted
intestinal sac method using the proximal 10 cm of intestine
as described earlier.44 Statistical analysis was by the Student’s
t test.46 Intestinal calcium transport is expressed as the
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 increase
in serum calcium must arise from bone and, hence, is a
determination of bone calcium mobilization.
2. Mea su r em en t of Cellu la r Differ en tia tion . Human
leukemia HL-60 cells (obtained from ATTC) were plated at 2
× 105 cells per plate and incubated in Eagle’s modified medium
as described previously.4c 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 micros-
copy using a hemacytometer. At least 200 cells were counted
in duplicate per determination. Percentage differentiation
represents the percentage of cells providing NBT reduction
appearance. The results were plotted on semilog paper, and

3378 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 16
Sicinski et al.
relative differentiation activities of the analogues were deter-
mined by comparison of the compound concentrations capable
of inducing 50% maturation according to the assay. This
method is described in detail elsewhere.4c The experiment was
repeated 3 times, and the results are reported as the mean (
SD.
2. Dock in g of An a logu es to th e Liga n d Bin d in g P ock et
of th e VDR. Docking simulations of four analogues (6a -9a ),
whose synthesis is described in this work, were performed by
FlexiDock software from TRIPOS50 which employs genetic
algorithms for the search of conformational space of the ligand
with respect to the given structure of the receptor’s binding
site. Because FlexiDock requires an approximate starting
position of the ligand to be provided, the docking procedures
were carried out several times for each analogue, starting from
arbitrarily chosen emplacement of the ligand with respect to
the binding pocket of the receptor. Internal rotations around
the single bonds (ring OH groups and side chain) of the ligands
were allowed during the simulations. Several simulations (of
50 000 steps each) were performed with different initial
positioning of the compound in the vicinity of the pocket and
converged to approximately the same structure within the
binding site. For final consideration, the structures of the
lowest conformational energy of the ligand-receptor complex
were selected.
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.47 It was diluted, and 0.1 mg of
protein (200 fmol of binding activity) in 100 µL was used in
each tube. 1R,25-(OH)2[26,27-3H]D3 (10 000 cpm) was added
in 2.0 µL of ethanol. To this mixture 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) was added. The sample was vortexed at 5 min intervals
for a total of 15 min on ice. The hydroxyapatite was then
washed 3 times by adding 0.5 mL of TE 5% Triton X-100,
centrifuging at 200g for 5 min, and aspirating the supernatant.
The radioactivity bound to the hydroxyapatite was determined
by liquid scintillation counting in Bio-Safe II scintillation fluid.
The values are plotted versus concentration of analogue or
standard. Each value represents triplicate values. The dis-
placement experiments were carried out in triplicate on two
different occasions.
Coordinates of the molecular models discussed in this work
can be found on our home page (http://biocomp.chem.uw.
edu.pl).
Ack n ow led gm en t. The authors are grateful for the
generous amount of computer time granted to them by
the Interdisciplinary Center for Mathematical and
Computational Modeling of Warsaw University (ICM).
We acknowledge the assistance of Rowland Randall in
recording the mass spectra. The work was supported
in part by funds from the Wisconsin Alumni Research
Foundation and the National Foundation for Cancer
Research.
Molecu la r Mod elin g. 1. Mod elin g of th e VDR Th r ee-
Dim en sion a l Str u ctu r e. The computations in this work
involve several steps. In the crucial one, we employed lattice
modeling (SICHO method) to generate the full-length model
of the ligand binding domain of the vitamin D receptor.
Homologous parts of the receptor (residues 118-163 and 207-
423) were built using the crystal structure of the hVDR
truncated mutant (118-425, ∆[165-215]) as the template for
traditional comparative modeling. Such a model is lacking the
highly nonhomologous insertion domain of the protein (en-
compassing residues 164-207). It is well-known that such big
gaps in protein structures cannot be reasonably filled by using
conventional comparative modeling tools.34,35,49 It is also
impractical (due to the extreme computational cost) to as-
semble these large protein fragments by means of standard
molecular dynamics simulations. For these reasons, we em-
ployed the recently developed lattice model for protein folding
and distant homology modeling.35 This method used the
simplified SICHO protein models and a set of statistical
potentials for efficient searching of protein conformational
space. The SICHO model enables simulations of the entire
folding process of small proteins, large-scale relaxation of poor-
quality models originated from sparse alignments (or thread-
ing alignments), and “docking” of relatively large protein
fragments to the partially determined protein structures.49 The
last is exactly the case of the present application. Thus, in the
first stage, we built the homologous part of the structure using
rather standard procedures described previously.34 The crystal
structure of the holo hVDR deletion mutant (118-425, ∆[165-
215]) was employed as a template for comparative modeling
of this well-defined part of the structure. The nonhomologous
part (residues 165-207) of the full-length model (residues
118-423) of rVDR-LBD was assembled on the scaffold of this
Su p p or tin g In for m a tion Ava ila ble: Purity criteria for
target vitamin D compounds; spectral data of the synthesized
compounds 6a ,b-9a ,b, 12-18, and 20a ,b-21a ,b; and figures
with either the competitive binding curves or dose-response
curves derived from the cellular differentiation assay of the
vitamin D analogues 6a ,b-9a ,b. This material is available free
of charge via the Internet at http://pubs.acs.org.
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