Development of analogues of 1alpha,25-dihydroxyvitamin D3 with biased side chain orientation: methylated des-C,D-homo analogues.

Article abstract of DOI:10.1002/1521-3765(20010119)7:2<520::AID-CHEM520>3.0.CO;2-5

The discovery that 1alpha,25-dihydroxyvitamin D3 is effective in the inhibition of cellular proliferation and in the induction of cellular differentiation has led to a search for analogues in which these activities and the classical calcemic activity of t

Full text of DOI:10.1002/1521-3765(20010119)7:2<520::AID-CHEM520>3.0.CO;2-5

FULL PAPER
Development of Analogues of 1a,25-Dihydroxyvitamin D3 with Biased Side
Chain Orientation: Methylated Des-C,D-Homo Analogues
Stefan GabrieÈls,[a] Dirk Van Haver,[a] Maurits Vandewalle,[a] Pierre De Clercq,*[a]
Annemieke Verstuyf,[b] and Roger Bouillon[b]
Abstract: The discovery that 1a,25-dihydroxyvitamin D3 is effective in the inhibition
of cellular proliferation and in the induction of cellular differentiation has led to a
search for analogues in which these activities and the classical calcemic activity of this
hormone are separated. In this context, the synthesis and biological evaluation are
reported of the three stereoisomeric CD-ring modified structural analogues in order
to enforce a particular and different orientation of the 25-hydroxylated side chain.
Comparison of the results of the biological evaluation and conformational analysis of
the side chain suggests one defined and ªactiveº geometry.
Keywords: structure ± activity rela-
tionships ´ terpenoids ´ vitamin D3
A-ring, our laboratory has focussed on the development of
analogues in recent years that vary in the structure of the
central CD-ring system.[11] A typical example is analogue 3,
the structure of which is characterized by the absence of the
six-membered C-ring and by the presence of an enlarged
D-ring, that is a formal 8,9-seco-9,11-bis-nor-15-homo deriv-
ative.[11f] For the sake of simplicity and clarity we will further
use ª6D analoguesº as descriptive term. Furthermore, the
carbon atoms of the modified vitamin D skeleton will be
referred to according to the conventional steroid numbering.
Introduction
The last decade has witnessed a very active search for
analogues of 1a,25-dihydroxyvitamin D3 (1, further abbrevi-
ated as 1,25(OH)2D3), the hormonally active form of vita-
min D.[1] Next to its classical calciotropic activity,[2]
1,25(OH)2D3 (1) has been shown to possess immunosuppres-
sive activity,[3] to inhibit cellular proliferation, and induce
cellular differentiation.[4] Its therapeutic value in the treat-
ment of certain cancers and skin diseases is, however, limited
since effective doses provoke calcemic side effects such as
hypercalcemia, hypercaliuria, and bone decalcification. This
has stimulated the development of analogues of the natural
hormone in which the calcemic activity and the antiprolifer-
ative and/or prodifferentiating activities are separated.[5] In
this context various successful structural modifications have
already been introduced, such as 19-nor,[6] 22-oxa,[7] 23-yne,[8]
20-epi (2),[9] 1-hydroxymethyl[10] derivatives, and combina-
tions thereof. Whereas the above modifications are located in
the flexible parts of the molecule, that is the side chain and the
[a] Prof. Dr. P. De Clercq, S. GabrieÈls, D. Van Haver,
Prof. Dr. M. Vandewalle
Laboratory for Organic Synthesis
Department of Organic Chemistry, Ghent University
Krijgslaan 281, 9000 Gent (Belgium)
Fax : (‡ 32)9-2644998
E-mail: pierre.declercq@rug.ac.be
[b] Dr. A. Verstuyf, Prof. Dr. R. Bouillon
Laboratory of Experimental Medicine and Endocrinology
Catholic University Leuven, Gasthuisberg
Herestraat 49, 3000 Leuven (Belgium)
520
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520±532
The comparison of some relevant biological activities for
compounds 1 ± 3 shown in Table 1 illustrates the possibility of
discrimination of the various actions of vitamin D. It is hereby
striking how with similar affinities for the vitamin D receptor
(VDR), the 20-epimer 2 is several orders of magnitude more
potent than the natural hormone 1 both in prodifferentiating
and calcemic activity,[12] whereas 6D analogue 3 is much less
calcemic. The higher activity of 2 compared with 1 can be
associated with different orientations of the side chain (see
below).
conformation(s) is (are) shown on the basis of: 1) a chair
conformation for the six-membered D-ring possessing the two
larger substituents on C14 and C17 in equatorial position; 2)
the four-carbon C17-C20-C22-C23 chain adopting conforma-
tions in which steric interactions are minimized, in particular
in which syn-pentane interactions are avoided.[13] Since all
segments, cyclic and acyclic, are considered to adopt mini-
mum energy staggered conformations, relevant geometries
can be generated on a diammond lattice (shown as dotted
lines).[14] Black circles further indicate positions on the lattice
that, if occupied by a carbon atom of the backbone chain, that
is C22 in relation to the (C17, C20)-bond rotation and C23
(shown as R) in relation to the (C20, C22)-bond rotation,
would generate a destabilizing syn-pentane interaction; the
penalty of occupying such position has been evaluated at 7 ±
9 kJmolꢀ1.[15] On this exclusion basis, and referring to the
torsion angles C13-C17-C20-C22/C17-C20-C22-C23, the pre-
ferred side chain conformations correspond in the a-series to a
Table 1. Selected biological activities of 1 ± 3.[a]
Binding
Differentiation Calcium
Entry VDR(pig) DBP (human) HL-60
Ca serum (mice)
1
2
3
100
88
125
100
0.2
80
100
3000
90
100
800
4
‡
‡
(g /a)-conformation,[16] in the b-series to a (a/a)- or (a/g )-
[a] The activities are presented as relative values, the reference value of
1,25(OH)2D3 (1) being defined as 100%. Further details about the
methodology are given in the Experimental Section.
conformation, and in the c-series to a (gꢀ/a)- or (gꢀ/gꢀ)-
‡
conformation, where a, g , and gꢀ designate the three
possible staggered geometries that correspond to backbone
dihedral angles of 1808, ‡608, and ꢀ608, respectively. The
reason why these three particular side chain orientations were
selected will become clear in the Discussion.
Results and Discussion
The present study fits into a project aiming at the develop-
ment of analogues possessing side chains with biased spatial
orientations. In particular 6D-analogues 4 are presented here
so that, depending on the relative orientation of the methyl
substituents at C13 and C16, the side chain at C17 would
adopt different and distinct conformations. The three selected
configurations a, b, and c constrain the mobility in the
segment C17-C20-C22 in a way that is further illustrated in
Figure 1. For each of these configurations the preferred
Synthesis: The enantioselective preparation of analogues 4a,
4b, and 4c is outlined in the Schemes 1, 3, 5, and 6. In
Scheme 1 the conversion of the chiral pool monoterpene (R)-
(ꢀ)-carvone (5) into the three cyclohexanone derivatives 7a,
7b, and 7c is described in which the desired stereochemistry at
C13, C14, and C16 is established. The introduction of the
ethoxycarbonylmethyl substituent (cf. 13), which will further
serve as a handle for the construction of the desired side chain,
with concomittant formation of the required C17 stereo-
center, is outlined in Scheme 3.
Figure 1. Preferred orientation of the side chain in the three series a, b, and
c following the analysis of diamond-lattice type conformations in the
segment C13-C17-C20-C22; black circles correspond to positions that, if
occupied by a backbone carbon atom, would generate a syn-pentane
interaction.
The final conversion of esters 13 into the corresponding
analogues 4a, 4b, and 4c, following a common sequence in the
three series a, b, and c, is shown in Schemes 5 and 6. Key-
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Table 2. Relevant 1H NMR spectral data of diastereomeric ketones 7a, 7b,
and 7c.[a]
H13
H16
Entry
d
J(13,14) J(13,Me) SJexptl
d
J(16,15) J(16,Me) SJexptl
7a
7b
7c
2.37 12.1
2.57 10.3
2.70 ꢁ 5
6.5
6.7
7.3
29.6 2.42 12.8, 5.6 6.4
29.2 2.57 [b] 7.2
26.5 2.59 12.7, 6.4 6.4
37.1
30.3
38.2
[a] Determined by double irradiation of methyl groups located on C13 and
C16; coupling constants [Hz]. [b] Could not be determined due to
overlapping signals.
methyl groups in the three series involving chair cyclohex-
anones with the large 2-propenyl group in equatorial position
is substantiated by the magnitude of the vicinal coupling
constants (individual or as a sum) of H13 and H16, with the
most indicative coupling with the methyl substituent (>7.0 Hz
for an axial Me,<7.0 Hz for an equatorial Me).[21] Presumably,
the preferred formation of ketones 7a (from 6a) and 7c (from
6b) is the result of kinetic control.[22] In particular proton
addition to the enol (or enolate anion) that is obtained upon
the conjugate hydride addition determines the configuration
at C16. In both cases the stereoelectronically preferred
perpendicular addition would occur from the b-face. Indeed,
as shown in Scheme 2, in the case of 6a unhindred b-
protonation at C16 can occur on the preferred half-chair enol
conformation; in the case of 6b perpendicular protonation on
the more stable half-chair enol conformation would also occur
from the b-face. In any event, the followed reaction pathway
allows for obtaining the three required cyclohexanones in
sufficient amount for further conversion to the desired
analogues.
Scheme 1. Synthesis of diastereomeric ketones 7a ± c. a) LDA, THF; MeI,
DMPU. b) NaOMe, MeOH/THF. c) Na2S2O4, Adogen 464, NaHCO3,
toluene/H2O.
features of the latter sequence involve i) the attachment of the
remaining portion of the side chain through nucleophilic
alkynyl substitution followed by hydrogenation of the triple
bond (cf. 18 to 20 in Scheme 5), and ii) the attachment of the
seco-B,A-ring part (cf. 25) in a Horner± Wittig reaction of
aldehyde 24 (Scheme 6).[17] We further note that out of the
eleven steps of this sequence (13 to 4), six (!) are somehow
involved in the conversion of the 2-propenyl group into the
required aldehyde function. These steps became necessary as
a consequence of: i) the deliberate choice of introducing the
side chain via the above alkynyl pathway since thus 22-yne
analogues also become available; ii) the unexpected obser-
vation that the Horner coupling failed at the methyl ketone
stage (cf. 21).
The introduction of the methyl group at C13 involves a
classical alkylation reaction on (R)-(ꢀ)-carvone (5) which led
to a separable 1:1 mixture of 6a and 6b.[18] The configurational
assignment of both isomers at this stage follows from the
observation that isomerization in base (sodium methoxi-
de)[18b] leads to an equilibrium mixture in favor of the more
stable isomer 6a (6a:6b ˆ 9:1).[19] Further conjugate reduc-
tion of 6a and 6b with sodium dithionite under phase-transfer
conditions led to mixtures of ketones 7 in which the
diastereomers 7a and 7c, respectively, predominate.[20] The
confirmation of the configuration of the three required
ketones 7a, 7b, and 7c convincingly follows from inspection
of the 1H NMR spectral data; the most relevant of which are
summarized in Table 2. The relative disposition of the two
Scheme 2. Possible explanation for the preferred formation of 7a and 7c
upon conjugate reduction of 6a and 6b.
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1a,25-Dihydroxyvitamin D3 Analogues
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The introduction of the ethoxycarbonylmethyl substituent
with the required configuration at C17 (cf. 13) is performed in
two stages (Scheme 3): Attachment of the two-carbon chain
as a,b-unsaturated ester (8a, 11b, and 12c, respectively),
of 7b and 7c led directly to the desired unsaturated ester
derivatives in a single isomeric form, that is E-11b and Z-12c.
Again the configurational assignment of both compounds
rests on the detailed analysis of the relevant 1H NMR spectral
data shown in Table 3.[27] It is symptomatic how in both cases
the ester group has lodged itself in the open space that is
available through the axial arrangement of one of the methyl
groups.
Table 3. Relevant 1H NMR spectral data of diastereomeric unsaturated
esters 11b and 12c.[a]
H13
H16
Entry
d
J(13,Me)
SJexptl
d
J(16,Me)
SJexptl
11b
12c
2.42
4.39
6.5
7.3
30
26
4.16
2.44
7.2
6.5
[b]
37
[a] Determined through double irradiation of the methyl group located on
C13 and C16; coupling constants [Hz]. [b] Could not be determined due to
overlapping signals (COOCH2CH3).
The subsequent conjugate reduction of the a,b-unsaturated
esters 13 proved quite instructive. Indeed, whereas in each
series the lithium/liquid ammonia reduction led selectively to
the more stable diastereomer, that is 13a, 13b, and 13c, the
magnesium in methanol reduction afforded a mixture of 13
and 14 in each case as the two possible stereoisomers at C17
(Scheme 4). The latter observations are in line with the result
Scheme 3. Synthesis of diastereomeric esters 13a ± c. a) Ethoxyethynyl-
MgBr, toluene/THF; dil. H2SO4, THF. b) AcCl, PhNMe2, CHCl3.
c) KOtBu, tBuOH. d) Li, liq. NH3, tBuOH, Et2O.
Scheme 4. Conjugate reduction of unsaturated esters 8a, 11b, and 12c
with magnesium in methanol.
that has been obtained in the past upon reduction of a similar
substrate.[23] This repeated lack of stereoselectivity somehow
throws a shadow on the general usefulness of the magnesium/
methanol conjugate reduction reaction of a,b-unsaturated
esters.[28] On the other hand, the availability of the C17
isomeric esters 14 in the three series a, b, and c, even as
inseparable mixtures with the corresponding isomers 13,
turned out to be very useful in the context of the structural
determination of 13a, 13b, and 13c. Careful analysis of the 1H
COSY NMR spectra of the mixtures of 13 and 14 allowed the
distinction between the two C17 isomeric series, in particular
on the basis of the magnitude of the sum of the vicinal
followed by dissolving metal reduction to afford esters 13a,
13b and 13c, respectively.[23] Whereas the direct Peterson
olefination proved unsuccessful,[24] a two-stage process in-
volving addition of the Grignard derivative derived from
ethoxyethyne, followed by sulfuric acid hydrolysis, afforded
the required unsaturated derivatives.[25] In the case of 7a this
reaction led to a separable 1:1 mixture of the desired
unsaturated ester 8a (as an unseparable E,Z-mixture) and
the tertiary alcohol 9a. Elimination of the latter through
subsequent formation of the corresponding acetate 10a led to
a further portion of 8a.[26] In contrast to the a-series, reaction
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coupling constant values of H17, which turns out to be larger
in the isomers 13 which are characterized by the preferred cis-
diequatorial orientation of the two larger substituents at C14
an C17 (Figure 2). The similarity between the signals observed
for the CH2COOEt moiety in the b- and c-series is striking
and fully in accord with the pseudo-enantiomeric relationship
that one may identify between the two series.
Scheme 5. Synthesis of ketone 21a. a) LAH, THF. b) O3, CH2Cl2/MeOH,
ꢀ408C. c) HO(CH)2OH, HC(OMe)3, pTsOH. d) pTsCl, CH2Cl2, Et3N,
DMAP. e) NaH, DMSO; OEE-protected 2-methyl-3-butyn-2-ol, DMSO.
f) 5% Rh/Al2O3, H2, EtOAc. g) PPTS, acetone/H2O.
Figure 2. Relevant 1H NMR spectral data of the diastereomeric esters
13a ± c and 14a ± c: chemical shifts (d) and vicinal coupling constant values
in parentheses [Hz]; assignments aided by H,H-COSY. R ˆ isopropenyl.
The sequence followed for the further conversion of esters
13a, 13b, and 13c into the corresponding ketones 21a, 21b,
and 21c is described in detail for the a-series (Scheme 5).
After reduction of ester 13a to the corresponding alcohol 15a,
the oxidative cleavage (ozone in dichloromethane/metha-
nol)[29] of the propenyl double bond led to ketone 16a. After
protection of the carbonyl through acetalisation to 17a and
conversion of the alcohol to the corresponding tosylate 18a,
substitution with the sodium salt of 2-methyl-3-butyn-2-ol,
protected as ethoxyethyl ether, afforded alkyne 19a in good
yields.[30] Catalytic hydrogenation to 20a followed by acid
hydrolysis led to methyl ketone 21a.
Scheme 6. Synthesis of vitamin D analogues 4a, 4b, and 4c. a) NaBr,
MeOH, Pt-electrode, 20 ± 30 V. b) LAH, THF. c) PCC, CH2Cl2. d) nBuLi,
25, THF; TBAF.
The final conversion of the methyl ketones 21a, 21b, and
21c into the vitamin D analogues 4a, 4b, and 4c is outlined in
Scheme 6. The transformation of ketone 21 into the required
aldehyde 24 for subsequent attachment of the seco-B,A-ring
portion of the vitamin D molecule involved three steps: 1) the
electrochemical oxidation of 21 into ester 22,[31] 2) lithium
aluminumhydride reduction to 23, and 3) oxidation with
pyridinium chlorochromate supported on alumina to 24.[32]
The first step is the electrochemical variant of the classical
bromoform reaction: two platinum electrodes are immersed
in a solution of ketone 21 and sodium bromide as the
electrolyte in methanol and submitted to a 20 ± 30 V tension.
For reasons that remain unclear, in the a-series the yield was
deceivingly low: 28% for 22a, compared with 46% for 22b,
and 76% for 22c. As shown in Table 4 analysis of the signal
observed for H14 in the 1H NMR spectrum of the ester
derivatives 22 allows for the confirmation of the configura-
tional assignment in the series of three. Finally, treatment of
the aldehydes 24a, 24b, and 24c with the anion derived from
the known phosphine oxide 25,[33] followed by in situ silylether
deprotection with tetrabutylammonium fluoride (TBAF), led
to the analogues 4a, 4b, and 4c in fair yields.
Following the above sequence isomerization at C14 is in
principle possible at several stages (cf. formation of 16, 17, 21,
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1a,25-Dihydroxyvitamin D3 Analogues
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22, 24, and even 4). Nevertheless it is assumed that, at least
from 21 onward, the more stable diastereomer will predom-
inate. As expected, calculations performed on the methyl
ester 22 indicate the shown (14R)-stereoisomer to be the more
stable epimer in each series.[34] This is further substantiated by
the intracellular vitamin D receptor (VDR) so as to regulate
gene transcription and synthesis of new proteins that are more
directly responsible for the biological response. In view of this
mechanism the geometry of the VDR-1,25(OH)2D3 complex
is crucial. Until recently,[36] no precise information about the
active shape of the hormone was known because of problems
encountered in obtaining a detailed crystal structure of the
liganded nuclear receptor (see below).[37] Therefore indirect
means had to be developed. The determination of the active
shape of the vitamin D hormone is intrinsically difficult
because of its flexible nature. Indeed, flexible ligands are
known to undergo substantial distortions away from their
preferred geometry in order to achieve optimal binding with
the receptor.[38] Since a greater preorganization of the active
geometry is expected to result in enhanced binding, it is
logical to conceive analogues that are characterized by
reduced mobility in the flexible parts of the molecule. With
regard to the side chain this may consist in constraining one or
1
the H NMR spectral data shown in Table 4.
Table 4. Relevant 1H NMR spectral data of diastereomeric esters 22a ± c (R ˆ
4-hydroxy-4-methylpentyl).[a]
H14
H13
d(Me) J(H13,Me) d(Me) J(H16,Me)
2.03 td 11.6, 11.6, 3.1 0.81[b] 6.4 0.89[b] 6.4
H16
Entry
d
m
Jvic
22a
22b
22c
2.00 td 11.5, 11.5, 3.9 0.82
2.41 dt 11.0, 4.8, 4.19 0.69
6.3
7.1
0.85
0.85
7.1
6.4
ꢀ
several of the rotatable carbon carbon bonds along the C17 ±
C25 chain. Whereas this goal can be achieved by unsaturated
moieties within the side chain,[8] a more subtle way may
consist in changing the substitution pattern in this part of the
molecule. The present work has been performed in this
particular context.
[a] Coupling constants in Hz. [b] Data of the methyl ketone 21a.
Biological evaluation: The biological evaluation of analogues
4a ± c includes the determination of 1) the binding affinity for
the porcine intestinal VDR, 2) the antiproliferative activity in
vitro on breast cancer MCF-7 cell, 3) the cell-differentiating
activity in vitro on a leukemic HL-60 cell line, and 4) the
calcemic activity in vivo in vitamin D-replete normal NMRI
mice. Results are shown in Table 5.
Central in this and related work is the use of conformational
maps to describe the preferred geometries of the side chain.
The concept of dot maps in this area was first introduced by
Okamura and Midland.[39] In this approach force field
calculations are performed so as to generate within a given
energy window all possible local minimum energy conforma-
tions that the side chain may adopt. The orientation in space
of each found conformation is further defined by a dot that
corresponds to the position of the 25-oxygen atom in that
particular conformation. Subsequently, volume maps have
been used in our laboratory in order to optimize the visual-
ization aspect of the procedure.[40] Volume maps correspond-
ing to the side chain conformations of 4a ± c are presented in
Figure 3. Note that these have been generated for model
derivatives that lack the A-ring. Three different views are
shown: next to the classical top and front views, a view
analogous to the one used in Figure 1 has been included. From
inspection of Figure 1 it is readily apparent that with each
configuration a, b, and c fit a specific and different side chain
orientation. To a fair extent the latter also correspond nicely
with those deduced via the exclusion procedure that is
illustrated in Figure 1. Prior to this work four conformation-
ally restricted 22-methyl substituted analogues of
1a,25(OH)2D3 (1), diastereomeric at C20 and C22, have been
studied by Yamada et al., with the aim to study the three-
dimensional structure of vitamin D that is involved in the
binding to the receptor.[41] Interestingly, one among those was
found to possess a markedly higher binding affinity for the
VDR; moreover, the same diastereomer was substantially
more potent than the natural hormone 1 in inducing differ-
entiation of HL-60 cells. In a more recent study dealing with
structure ± function relationships related to the orientation of
the side chain, the same group identified particular regions in
which the preferred orientation of the 25-hydroxy group
would correspond to the increased cell-differentiating
Table 5. Selected biological activities of 4a ± c.[a]
Binding
Cell differentiation
and proliferation
Calcium
Entry
VDR(pig)
HL-60
MCF-7
Ca serum (mice)
1
100
20
2
100
90
9
100
200
30
100
4
< 0.25
< 0.25
4a
4b
4c
2
8
70
[a] The activities are presented as relative values, the reference value of
1,25(OH)2D3 (1) being defined as 100%. Further details about the
methodology are given in the Experimental Section.
The 6D-analogue 4a displayed 20% of the VDR affinity
compared with 1,25(OH)2D3 (100% binding, Table 5). The
two epimers 4b and 4c demonstrated only 2% of the affinity
for the VDR. The 6D-analogue 4a has equipotent prodiffer-
entiating activity when compared with the activity of
1,25(OH)2D3 on HL-60 cells while this analogue was two
times more potent in the inhibition of the proliferation of
MCF-7 cells. Epimers 4b and 4c were less potent in the
inhibition of the MCF-7 cell proliferation or the stimulation of
the HL-60 cell differentiation compared with 1,25(OH)2D3
whereas both analogues had poor calcemic effects in vivo
(<0.25% compared with 1,25(OH)2D3).
Conformational analysis: Vitamin D activity is normally
expressed via a genomic pathway:[35] The hormone binds with
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P. De Clercq et al.
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lowest possible mole fraction
of the side chain conformations
of the less active analogue, but
at the same time contains the
highest possible mole fraction
of the conformations of the
more active of the pair.[43] The
relative activity volume shown
was generated on the basis of
the conformational profiles of
the most and least active dia-
stereomers among the four 22-
methyl substituted analogues
developed by Yamada. The ap-
plied procedure has been de-
scribed in detail.[40] Also includ-
ed in Figure 4 is the location of
the 25-hydroxy group (green)
of the conformation of the nat-
ural ligand when bound to the
receptor. This important infor-
mation has become recently
available through the high-res-
olution crystal structure of the
complex between the VDR li-
gand binding domain and
1,25(OH)2D3.[36] This study
shows the three-dimensional
arrangement of the ligand-
binding
pocket
around
Figure 3. Volume maps representing the conformational behavior of the side chain of truncated models of 6D
analogues 4a, 4b, and 4c (middle, top and bottom line, respectively) in three different viewing directions: top
(left), front (middle), and side (right). Colored spheres indicate positions of O25; total energy window:
1,25(OH)2D3 and reveals in
particular that the elongated
ligand occupies only 56% of
20 kJmolꢀ1
.
the accessible volume of the
potency.[42] Following this active space group concept five
main regions designated as A, G, EG, EA, and F were
identified. With this study a correlation was proposed
between the cell-differentiating potency of numerous ana-
logues and the conformational space occupied by the
vitamin D side chain. As an important result it was observed
that the preferred side chain orientations of almost all potent
analogues were distributed
VDR cavity. Obviously, the results of these different con-
formational studies shown in Figure 4 are overlaid so as to
allow for a relative spatial comparison.
Finally, the side chain mappings corresponding to the
analogues 4a, 4b, and 4c following Yamadaꢁs view are shown
in Figure 5, also including the above relative activity volume.
The occupation of this volume by the 25-hydroxy group of the
among
the
EA
region.
These different regions are vi-
sualized in Figure 4. Note how
within the same representation
are combined the side chain
mappings of 1a,25(OH)2D3
(yellow) and 20-epimer 2 (blue).
Further a superposition with
the so-called relative activity
volume (red) is shown in this
representation. The latter is
generated so that, among a pair
of epimeric analogues possess-
ing very different activities
(ideally a very active and an
inactive compound), a sphere
is created that contains the
Figure 4. Volume maps representing the conformational behavior of the side chain of 1,25(OH)2D3 (1) (yellow)
and its 20-epimer (2) (cyan) with the indicated regions A, G, EG, EA, and F following ref. [41]; the viewing
direction is given in the stereoscopic view. Relative activity volume (red) following ref. [40]. Position of 25-oxygen
in receptor-bound conformation (green) according to ref. [36].
526
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0947-6539/01/0702-0526 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 2

1a,25-Dihydroxyvitamin D3 Analogues
520±532
observations certainly warrant the continuation of the search
for analogues that would feature an even more pronounced
discrimination in activity.
Experimental Section
Synthesis: Thin-layer chromatography was performed on Merck silica gel
60F-254 TLC plates. All products were purified by flash chromatography
(Merck silica gel 60F254) or HPLC: Waters 4000, Kontron 420/422. [a]D20
(CHCl3): Perkin ± Elmer 241. IR (NaBr): Perkin ± Elmer 1600 series.
1H NMR (CDCl3): 500 MHz, Bruker AN-500 (internal TMS as reference).
13C NMR (CDCl3): 50 MHz, Varian Gemini-200 (with DEPT program).
MS: Finnigan 4000 or Hewlett ± Packard 5988A.
Conjugate reduction of (5R,6S)-2,6-dimethyl-5-isopropenyl-2-cyclohexe-
none (6a) and (5R,6R)-2,6-dimethyl-5-isopropenyl-2-cyclohexenone (6b):
A mixture of cyclohexenone 6a (2.91 g, 17.72 mmol), sodium bicarbonate
(26.8 g), and phase-transfer catalyst (Adogen 464, 0.96 g, 5.31 mmol) in
toluene (220 mL) and water (220 mL) was stirred vigorously under
nitrogen. Sodium dithionite (27.76 g, 159.45 mmol) was added and the
mixture heated under reflux for 2 h. After cooling, the aqueous layer was
separated and extracted with diethyl ether. The combined organic extracts
were washed with water, dried, and the solvent removed in vacuo. After
column chromatography on silica gel and further separation by HPLC
(hexane/diethyl ether 85:15) ketones 7a (2.35 g, 80%) and 7b (0.52 g,
18%) were obtained as colorless oils. In exactly the same way the reduction
of cyclohexenone 6b afforded ketone 7c (70% isolated yield).
Compound 7a: Rf ˆ 0.43 (pentane/acetone 98:2); [a]2D0 ˆ ꢀ2.5 (c ˆ 0.85,
CHCl3); IR (NaBr): nÄ ˆ 2927, 1712, 1644, 1454, 1376, 1317, 1180, 1132, 980,
946, 892 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆ 4.78 (m, 1H), 4.75 (m, 1H),
2.42 (ddq, 1H, J ˆ 12.8, 5.6, 6.4 Hz), 2.37 (dq, 1H, J ˆ 12.1 Hz, 6.5 Hz),
ꢁ2.08 (m, 2H), 1.85 (qd, 1H, J ˆ 12.9, 3.8 Hz), 1.73 (ABq, 1H, J ˆ 13.5,
3.4 Hz), 1.70 (s, 3H), 1.38 (ABq, 1H, J ˆ 13.1, 3.8 Hz), 1.02 (d, 3H, J ˆ
6.4 Hz), 0.90 (d, 3H, J ˆ 6.5 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ
ˆ
ˆ
ˆ
214.0 (C O), 146.2 (C ), 112.1 (CH2 ), 55.6 (CH), 47.5 (CH), 45.1 (CH),
35.5 (CH2), 31.4 (CH2), 18.0 (CH3), 14.6 (CH3), 11.9 (CH3); MS: m/z: (%):
‡
166 [M] , 164 (100), 149 (32), 135 (23), 121 (28), 107 (37), 91 (43), 77 (27),
69 (46), 43 (64).
Figure 5. Volume maps representing the conformational behavior of the
side chain of 6D analogues 4a (middle), 4b (top), and 4c (bottom) with
inclusion of the relative activity volume (red) according to ref. [40];
direction of view similar to the one used in Figure 4.
Compound 7b: Rf ˆ 0.40 (pentane/acetone 98:2); [a]2D0 ˆ ‡92.7 (c ˆ 1.03,
CHCl3); IR (NaBr): nÄ ˆ 2933, 1708, 1646, 1456, 1374, 1231, 959, 892 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ 4.79 (m, 1H), 4.72 (brs, 1H), 2.57 (dq, 1H,
J ˆ 10.3, 6.7 Hz), 2.57 (m, 1H), 2.13 (td, 1H, J ˆ 10.2, 4.0 Hz), ꢁ1.90 (m,
2H), 1.73 ± 1.57 (m, 2H), 1.71 (s, 3H), 1.18 (d, 3H, J ˆ 7.2 Hz), 0.96 (d, 3H,
13
ˆ
J ˆ 6.7 Hz); C NMR/DEPT (50 MHz, CDCl3): d ˆ 216.4 (C O), 145.9
ˆ
ˆ
side chain was calculated to correspond to 63%, 6%, and 5%
for 4a, 4b, and 4c. Interestingly the biological activity of the
6D-analogue 3 is quite similar to the one observed for 4a, in
particular the differentiation of HL-60 cells and calcemic
activity are identical.[11g] Furthermore, the conformational
profile of the side chain 3 is analogous to the one calculated
for 4a; in particular the occupation of the same relative
activity volume by the 25-hydroxy group of 3 corresponds to
69%. This further confirms the existence of a (qualitative)
correlation between side chain orientation and cell-differ-
entiating potency.
(C ), 111.8 (CH2 ), 53.1 (CH), 43.4 (CH), 43.1 (CH), 31.0 (CH2), 25.0
‡
(CH2), 18.4 (CH3), 16.4 (CH3), 12.7 (CH3); MS: m/z (%): 166 (35) [M] , 151
(15), 139 (1), 137 (10), 123 (23), 109 (54), 95 (43), 81 (100), 67 (81), 55 (79),
41 (84).
Compound 7c: Rf ˆ 0.39 (pentane/acetone 98:2); [a]2D0 ˆ ꢀ1.2 (c ˆ 1.07,
CHCl3); 1H NMR (500 MHz, CDCl3): d ˆ 4.90 (s, 1H), 4.73 (s, 1H), 2.70
(m, 1H, SJ ˆ 26.5 Hz), 2.59 (ddq, 1H, J ˆ 12.7, 6.4, 6.4 Hz), 2.39 (m, 1H),
2.07 (m, 1H), 1.90 (qd, 1H, J ˆ 13.0, 3.7 Hz), 1.68 (m, 1H), 1.67 (s, 3H), 1.30
(qd, 1H, J ˆ 13.0, 3.7 Hz), 1.02 (d, 3H, J ˆ 6.5 Hz), 0.95 (d, 3H, J ˆ 7.3 Hz);
13
ˆ
ˆ
C NMR/DEPT (50 MHz, CDCl3): d ˆ 216.7 (C O), 145.2 (C ), 111.2
ˆ
(CH2 ), 48.3 (CH), 46.7 (CH), 40.0 (CH), 34.3 (CH2), 23.7 (CH2), 22.1
(CH3), 14.5 (CH3), 11.8 (CH3).
Ethyl
[(2S,3R,6R)-2,6-dimethyl-3-isopropenylcyclohexylidene]acetate
The structure of 4 is peculiar in that, when compared with
1,25(OH)2D3, it lacks several atoms such as one methyl group
at C13, the methyl group at C20, and a substantial part of the
C-ring and possesses two extra carbon atoms in the D-ring.
Yet, these important structural changes in the central part of
the molecule do not prevent binding to the receptor. Whereas
these changes may be responsible for the observed diminished
calcemic activity, it is interesting to note how the cell
differentiation activity can, in a first approximation, be
regulated by the orientation of the side chain. These
(8a): Ethoxyethyne (0.33 mL, 2.35 mmol) and toluene (2 mL) were added
08C to a solution of ethylmagnesium bromide (1.98 mL, 1.99 mmol) in
tetrahydrofuran (3 mL). After raising the temperature to 608C a solution of
ketone 7a (0.3 g, 1.80 mmol) in toluene (0.8 mmol) was added; the reaction
mixture was further heated under reflux for 2 h and then poured into ice-
water. The organic phase was separated after addition of ammonium
chloride and the aqueous phase was extracted with diethyl ether. The
organic phases were combined, washed with water, and concentrated in
vacuo. The obtained residue was further dissolved in tetrahydrofuran
(42 mL) and treated with 10% sulfuric acid (0.9 mL). After stirring for
10 h, the solution was concentrated in vacuo and the residue extracted with
diethyl ether. The combined ether phases were washed with water and
Chem. Eur. J. 2001, 7, No. 2
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0947-6539/01/0702-0527 $ 17.50+.50/0
527

P. De Clercq et al.
FULL PAPER
dried (MgSO4). After filtration and concentration in vacuo, the residue was
purified by column chromatography and HPLC (pentane/acetone 98:2 to
95:5) to afford ester 8a (0.189 g, 44%) and alcohol 9a (0.221 g, 48%).
(CH), 36.4 (CH2), 33.3 (CH), 32.6 (CH), 24.1 (CH2), 22.4 (CH3), 18.2 (CH3),
14.4 (CH3), 13.0 (CH3); MS: m/z (%): 236 (3) [M] , 221 (9), 207 (3), 193
(65), 165 (20), 154 (100), 121 (49), 107 (60), 81 (56), 69 (56), 41 (83).
‡
Metal reduction of the a,b-unsaturated esters 8a, 11b, and 12c with lithium
in liquid ammonia: A solution of ester 8a (0.435 g, 1.84 mmol) in diethyl
ether (11 mL) and tert-butanol (0.14 mL) was added dropwise to a solution
of lithium (0.037 g, 5.34 mmol) in liquid ammonia (100 mL, distilled from
sodium in the presence of FeCl3). After stirring for 20 min the reaction
mixture was quenched with ammonium chloride. After further stirring for
30 min, the ammonia was evaporated and the residue extracted with diethyl
ether. The organic phase was dried (MgSO4) and concentrated in vacuo.
Column chromatography and HPLC (hexane/ethyl acetate 96:4) afforded
ester 13a (0.408 g, 93%). In the same manner unsaturated esters 11b
(0.746 g) and 12c (0.650 g) afforded pure 13b (82%) and 13c (68%),
respectively.
Compound 8a (inseparable mixture of E/Z 55:45): Rf ˆ 0.33 (pentane/
diethyl ether 98:2); IR (NaBr): nÄ ˆ 2934, 1713, 1631, 1454, 1383, 1221, 1195,
1166, 1150, 1099, 1037, 891, 869 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆ 5.68
(s, 1H), 5.65 (s, 1H), 4.78 (m, 2H), 4.70 (m, 2H); MS: m/z (%): 236 (3)
[M] , 221 (4), 207 (3), 193 (75), 165 (30), 154 (31), 121 (38), 107 (34), 81
(46), 77 (46), 41 (100); C15H24O2 (236.35): calcd C 76.2, H 10.2; found C
76.15, H 10.23.
‡
Compound 9a: Rf ˆ 0.26 (pentane/diethyl ether 92:8); [a]2D0 ˆ ‡22.2 (c ˆ
1.19, CHCl3); IR (NaBr): nÄ ˆ 3550, 2927, 1715, 1645, 1443, 1374, 1301, 1197,
1096, 1032, 966, 888 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆ 4.71 (brs, 1H),
4.70 (brs, 1H), ꢁ4.14 (m, 2H), 2.57 (s, 2H), 2.12 (td, 1H, J ˆ 11.8, 3.5 Hz),
1.61 (s, 3H), 1.60 ± 1.35 (m, 7H), 1.27 (t, 3H, J ˆ 7.1 Hz), 0.95 (d, 3H, J ˆ
6.6 Hz), 0.86 (d, 3H, J ˆ 6.7 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ
Compound 13a: Rf ˆ 0.37 (pentane/diethyl ether 98:2); [a]2D0 ˆ ‡18.3 (c ˆ
ˆ
ˆ
ˆ
ꢀ
ꢀ
1.00, CHCl3); IR (NaBr): nÄ ˆ 2924, 1732, 1450, 1373, 1157, 1039, 885 cmꢀ1
;
172.5 (C O), 149.0 (C ), 111.2 (CH2 ), 74.6 (C O), 60.4 (CH2 O), 47.7
(CH), 41.3 (CH), 40.8 (CH2), 39.7 (CH), 31.8 (CH2), 30.0 (CH2), 18.4 (CH3),
1H NMR (500 MHz, CDCl3): d ˆ 4.68 (m, 2H), 4.12 (m, 2H), 2.42 (d, 2H,
J ˆ 4.5 Hz), ꢁ1.70 (m, 2H), 1.61 (s, 3H), 1.55 ± 1.18 (m, 5H), 1.25 (t, 3H,
‡
16.0 (CH3), 14.1 (CH3), 12.5 (CH3); MS: m/z (%): 254 (3) [M] , 236 (15),
J ˆ 7.1 Hz), 1.10 (m, 1H), 0.93 (d, 3H, J ˆ 6.4 Hz), 0.83 (d, 3H, J ˆ 6.4 Hz);
208 (8), 197 (19), 170 (23), 144 (44), 123 (26), 109 (36), 82 (56), 69 (84), 55
(100); C15H26O3 (254.37): calcd C 70.8, H 10.3; found C 70.72, H 10.28.
13
ˆ
ˆ
C NMR/DEPT (50 MHz, CDCl3): d ˆ 173.6 (C O), 149.4 (C ), 110.7
ˆ
(CH2 ),60.0 (CH2-O), 53.6 (CH), 47.5 (CH), 38.1 (CH), 36.6 (CH), 36.1
(CH2), 35.5 (CH2), 32.0 (CH2), 20.6 (CH3), 18.7 (CH3), 17.3 (CH3), 14.2
(CH3); MS: m/z (%): 238 (12) [M] , 223 (3), 195 (28), 192 (5), 164 (9), 150
Compound 10a: A solution of alcohol 9a (0.336 g, 1.32 mmol), N,N-
dimethylaniline (8.6 mL, 68.12 mmol), and acetyl chloride (1.86 mL,
26.22 mmol) in chloroform (13 mL) was heated at reflux for 24 h. The
resulting solution was diluted with water and acidified with hydrochloric
acid. After extraction with diethyl ether, the organic phase was dried
(MgSO4), filtered, and concentrated in vacuo. Column chromatography
and HPLC (pentane/acetone 99:1) afforded acetate 10a (0.279 g, 71%).
Rf ˆ 0.27 (pentane/acetone 99:1); [a]2D0 ˆ ‡28.3 (c ˆ 1.16, CHCl3); 1H NMR
(500 MHz, CDCl3): d ˆ 4.70 (m, 2H), 4.12 (m, 2H), 3.54 (AB, 1H, J ˆ
15.1 Hz), 3.44 (AB, 1H, J ˆ 15.1 Hz), 2.15 (td, 1H, J ˆ 11.6, 3.6 Hz), 2.04 (s,
3H), 1.93 (m, 1H), 1.84 (dq, 1H, J ˆ 11.3, 6.7 Hz), 1.60 (s, 3H), 1.56 ± 1.40
‡
(80), 135 (38), 109 (56), 95 (75), 82 (98), 67 (77), 41 (100); C15H26O2
(238.37): calcd C 75.6, H 11.0; found C 75.57, H 10.91.
Compound 13b: Rf ˆ 0.33 (pentane/diethyl ether 98:2); [a]2D0 ˆ ‡35.8 (c ˆ
0.93, CHCl3); IR (NaBr): nÄ ˆ 2929, 1737, 1644, 1452, 1382, 1253, 1207, 1160,
1126, 1034, 888 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆꢁ 4.69 (m, 2H), 4.12
(m, 2H), 2.49 (dd, 1H, J ˆ 14.8, 5.7 Hz), 2.10 (dd, 1H, J ˆ 14.8, 9.0 Hz), 1.87
(m, 1H), 1.77 (m, 1H, SJ ˆ 30 Hz), 1.80 ± 1.30 (m, 6H), 1.63 (s, 3H), 1.25 (t,
3H, J ˆ 7.1 Hz), 0.90 (d, 3H, J ˆ 7.2 Hz), 0.75 (d, 3H, J ˆ 6.5 Hz); 13C NMR/
ˆ
ˆ
ˆ
DEPT (50 MHz, CDCl3): d ˆ 173.9 (C O), 149.2 (C ), 110.9 (CH2 ), 60.1
(CH2-O), 53.9 (CH), 43.7 (CH), 37.5 (CH2), 32.9 (CH2), 32.4 (CH), 31.2
(CH), 26.2 (CH2), 18.7 (CH3), 16.9 (CH3), 14.2 (CH3), 12.6 (CH3); MS: m/z
(m, 4H), 1.24 (t, 3H, J ˆ 7.1 Hz), 1.02 (d, 3H, J ˆ 6.8 Hz), 0.90 (d, 3H, J ˆ
13
ˆ
ˆ
6.7 Hz); C NMR/DEPT (50 MHz, CDCl3): d ˆ 170.5 (C O), 148.4 (C ),
ˆ
111.4 (CH2 ), 88.0 (C-O), 60.1 (CH2-O), 47.8 (CH), 40.6 (CH), 39.1 (CH),
‡
(%): 238 (23) [M] , 209 (3), 195 (35), 175 (12), 150 (100), 127 (68), 109 (62),
37.9 (CH2), 31.5 (CH2), 39.9 (CH2), 22.2 (CH3), 18.1 (CH3), 17.2 (CH3), 14.1
(CH3), 13.6 (CH3); C17H28O4 (296.40): calcd C 68.9, H 9.5; found C 69.01, H
9.46.
95 (96), 67 (70), 41 (74); C15H26O2 (238.37): calcd C 75.6, H 11.0; found C
75.62, H 11.02.
Compound 13c: Rf ˆ 0.31 (pentane/diethyl ether 98:2); [a]2D0 ˆ ꢀ60.5 (c ˆ
Conversion of 10a to 8a:
A solution of the acetate 10a (0.241 g,
1.36, CHCl3); IR (NaBr): nÄ ˆ 2926, 1736, 1644, 1444, 1373, 1334, 1302, 1270,
0.813 mmol) in a 1m solution of potassium tert-butoxide in tert-butanol
(1 mL) was stirred for 24 h at 258C. After extraction with diethyl ether, the
organic phase was dried (MgSO4), filtered, and concentrated in vacuo.
HPLC (pentane/diethyl ether 98:2) afforded 8a (0.146 g, 76%).
1251, 1189, 1159, 1126, 1032, 887 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ
4.77 (m, 1H), 4.57 (s, 1H), 4.16 (q, 2H, J ˆ 7.1 Hz), 2.47 (dd, 1H, J ˆ 14.9,
5.9 Hz), 2.12 (dd, 1H, J ˆ 14.9, 8.7 Hz), 2.00 (m, 2H), 1.77 ± 1.00 (m, 6H),
1.67 (s, 3H), 1.25 (t, 3H, J ˆ 7.1 Hz), 0.84 (d, 3H, J ˆ 6.5 Hz), 0.61 (d, 3H,
Ethyl [(E,2S,3R,6S)-2,6-dimethyl-3-isopropenylcyclohexylidene]acetate
(11b): Treatment of ketone 7b (0.80 g) with the Grignard derivative derived
from ethoxyethyne, followed by acid hydrolysis, as described for the
conversion of 7a to 8a, afforded 11b (70%). Rf ˆ 0.36 (pentane/diethyl
ether 98:2); [a]2D0 ˆ ꢀ12.1 (c ˆ 0.97, CHCl3); IR (NaBr): nÄ ˆ 2931, 1716,
13
ˆ
J ˆ 7.1 Hz); C NMR/DEPT (50 MHz, CDCl3): d ˆ 174.0 (C O), 148.0
ˆ
ˆ
(C ), 109.5 (CH2 ), 60.2 (CH2-O), 48.2 (CH), 45.3 (CH), 37.6 (CH2), 35.8
(CH2), 33.5 (CH), 30.7 (CH), 24.1 (CH2), 22.4 (CH3), 19.0 (CH3), 14.3
‡
(CH3), 6.9 (CH3); MS: m/z (%): 238 (13) [M] , 208 (1), 195 (32), 169 (13),
150 (79), 135 (36), 109 (92), 82 (98), 67 (100), 55 (46); C15H26O2 (238.37):
calcd C 75.6, H 11.0; found C 75.43, H 11.19.
1639, 1458, 1376, 1300, 1243, 1186, 1157, 1038, 890 cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d ˆ 5.59 (s, 1H), 4.73 ± 4.70 (m, 2H), 4.16 (m, 1H),
Reduction of a,b-unsaturated esters 8a, 11b, and 12c: Magnesium powder
(0.012 g, 0.494 mmol) was added to a solution of the ester 8a (0.050 g,
0.212 mmol) in dry methanol (2 mL). After consumption of the magnesium
(ca. 45 min) a second portion was added. This process was repeated twice
(total amount Mg: 0.048 g, 1.976 mmol). After cooling of the mixture,
acetic acid (0.23 mL) was added, followed by water (1 mL). The reaction
mixture was extracted with diethyl ether. The combined organic phases
were washed with water and dried (MgSO4). Column chromatography and
HPLC (hexane/ethyl acetate 97:3) afforded a mixture of 13a and 14a (8:3,
84%). In the same way the reduction of 11b (0.050 g) and 12c (0.050 g)
afforded mixtures of 13b and 14b (1:1, 79%) and 13c and 14c (1:1, 77%),
respectively.
4.15 (m, 2H), 2.42 (m, 1H), ꢁ1.82 (m, 2H), 1.70 (s, 3H), 1.65 ± 1.42 (m, 3H),
1.29 (t, 3H, J ˆ 7.1 Hz), 1.16 (d, 3H, J ˆ 7.2 Hz), 0.92 (d, 3H, J ˆ 6.5 Hz);
13
ˆ
ˆ
C NMR/DEPT (50 MHz, CDCl3): d ˆ 170.2 (C O), 167.0 (C ), 148.0
ˆ
ˆ
ˆ
(C ), 111.5 (CH2 ), 111.3 (CH ), 59.5 (CH2-O), 55.4 (CH), 35.3 (CH), 32.5
(CH2), 30.8 (CH), 26.3 (CH2), 18.5 (CH3), 18.2 (CH3), 15.3 (CH3), 14.3
‡
(CH3); MS: m/z (%): 236 (3) [M] , 221 (5), 193 (90), 191 (19), 165 (21), 147
(24), 133 (26), 121 (28), 107 (35), 81 (48), 67 (46), 41 (100); C15H24O2
(236.35): calcd C 76.2, H 10.2; found C 76.09, H 10.17.
Ethyl [(Z,2R,3R,6R)-2,6-dimethyl-3-isopropenylcyclohexylidene]acetate
(12c): Treatment of ketone 7c (0.50 g) with the Grignard derivative derived
from ethoxyethyne, followed by acid hydrolysis, as described for the
conversion of 7a to 8a, afforded 12c (51%). Rf ˆ 0.33 (pentane/diethyl
ether 99:1); [a]2D0 ˆ ꢀ78.7 (c ˆ 1.14, CHCl3); IR (NaBr): nÄ ˆ 2933, 1716,
Relevant spectral data for 14a (as mixture with 13a): 1H NMR (500 MHz,
CDCl3): d ˆ 4.68 (m, 2H), 4.12 (m, 2H), 2.28 (quintet, 1H, J ˆ 4.5 Hz), 2.17
(d, 2H, J ˆ 5.5 Hz), 1.61 (s, 3H), 1.25 (t, 3H, J ˆ 7.2 Hz), 0.86 (d, 3H, J ˆ
6.9 Hz), 0.75 (d, 3H, J ˆ 6.7 Hz).
1637, 1458, 1374, 1201, 1152, 1037, 889 cmꢀ1; H NMR (500 MHz, CDCl3):
1
d ˆ 5.58 (s, 1H), 4.83 (m, 1H), 4.66 (s, 1H), 4.39 (qd, 1H, J ˆ 7.1, 4.4 Hz),
4.16 (m, 2H), 2.44 (1H, SJ ˆ 37 Hz), 2.08 (m, 1H), 1.91 (dq, 1H, J ˆ 12.8,
3.0 Hz), 1.78 (qd, 1H, J ˆ 13.1, 3.9 Hz), 1.76 (s, 3H), 1.56 (m, 1H), 1.29 (t,
For 14b (as a mixture with 13b): 1H NMR (500 MHz, CDCl3): d ˆ 4.70 (m,
2H), 4.12 (m, 2H), 2.37 (ABd, 1H, J ˆ 15.2, 5.4 Hz), 2.30 (ABd, 1H, J ˆ
15.2, 9.1 Hz), 1.63 (s, 3H), 1.25 (t, 3H, J ˆ 7.2 Hz), 1.04 (d, 3H, J ˆ 7.1 Hz),
0.77 (d, 3H, J ˆ 7.2 Hz).
3H, J ˆ 7.2 Hz), 1.11 (qd, 1H, J ˆ 12.8, 3.8 Hz), 1.03 (d, 3H, J ˆ 6.5 Hz), 0.87
13
ˆ
(d, 3H, J ˆ 7.3 Hz); C NMR/DEPT (50 MHz, CDCl3): d ˆ 170.8 (C O),
ˆ
ˆ
ˆ
ˆ
167.2 (C ), 147.3 (C ), 110.5 (CH ), 110.1 (CH2 ), 59.6 (CH2-O), 48.0
528
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0702-0528 $ 17.50+.50/0
Chem. Eur. J. 2001, 7, No. 2

1a,25-Dihydroxyvitamin D3 Analogues
520±532
For 14c (as a mixture with 13c): 1H NMR (500 MHz, CDCl3): d ˆ 4.77
(m, 1H), 4.57 (m, 1H), 4.14 (m, 2H), 2.42 (ABd, 1H, J ˆ 14.8, 5.2 Hz), 2.32
(ABd, 1H, J ˆ 14.8, 9.7 Hz), 1.66 (s, 3H), 1.25 (t, 3H, J ˆ 7.1 Hz), 0.84 (d,
3H, J ˆ 6.5 Hz), 0.80 (d, 3H, J ˆ 7.2 Hz).
2-[(1S,2R,3R,6R)-3-Isopropenyl-2,6-dimethylcyclohexyl] ethanol (15a): A
solution of ester 13a (0.354 g, 1.48 mmol) in THF (2 mL) was added at 08C
to a solution of lithium aluminumhydride (0.113 g, 2.98 mmol) in tetrahy-
drofuran (3 mL). After stirring for 3 h at 258C, the reaction mixture was
quenched at 08C by the successive addition of water (0.113 mL), 15%
aqueous hydroxide solution (0.113 mL), and water (0.340 mL). After
filtration over a silica gel pad, the filtrate was concentrated in vacuo.
Column chromatography and HPLC (pentane/acetone 9:1) afforded
further concentrated and the residue purified by column chromatography
and HPLC (pentane/acetone 9:1) to afford tosylate 18a (0.092 g, 97%).
Rf ˆ 0.36 (pentane/acetone 9:1); [a]2D0 ˆ ‡2.9 (c ˆ 0.98, CHCl3); IR (NaBr):
nÄ ˆ 2879, 1456, 1362, 1177, 1097, 1040, 954, 815, 668 cmꢀ1
;
1H NMR
(500 MHz, CDCl3): d ˆ 7.77 (d, 2H, J ˆ 8.2 Hz), 7.37 (d, 2H, J ˆ 8.1 Hz),
4.00 (t, 2H, J ˆ 7.9 Hz), 3.93 (q, 1H, J ˆ 6.5 Hz), 3.85 (m, 1H), 3.80 (m, 2H),
2.43 (s, 3H), 1.92 (m, 1H), 1.85 ± 0.90 (m, 8H), 1.15 (s, 3H), 0.92 (d, 3H, J ˆ
6.3 Hz), 0.80 (d, 3H, J ˆ 6.4 Hz), 0.60 (tt, 1H, J ˆ 10.6, 3.5 Hz); 13C NMR/
DEPT (50 MHz, CDCl3): d ˆ 144.6 (Ar-C), 133.2 (Ar-C), 129.7 (2  Ar-
CH), 127.9 (2 Â Ar-CH), 112.0 (O-C-O), 68.7 (CH2-O), 64.2 (CH2-O), 63.0
(CH2-O), 51.1 (CH), 47.4 (CH), 37.6 (CH), 36.0 (CH), 35.3 (CH2), 28.9
(CH2), 28.3 (CH2), 21.5 (CH3), 20.5 (CH3), 19.2 (CH3), 17.3 (CH3); MS: m/z
(%): 381 (1), 155 (1), 87 (100), 43 (13); C21H32O5S (396.54): calcd C 63.6, H
8.1; found C 63.45, H 8.32.
alcohol 15a (0.283 g, 97%). Rf ˆ 0.33 (pentane/acetone 9:1); [a]D20
ˆ
‡15.4, (c ˆ 1.04, CHCl3); IR (NaBr): nÄ ˆ 3336, 2921, 1447, 1376, 1042,
886 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆꢁ 4.67 (m, 2H), 3.64 (t, 2H, J ˆ
7.9 Hz), 1.76 (td, 2H, J ˆ 8.0, 3.7 Hz), ꢁ1.68 (m, 2H), 1.61 (s, 3H), 1.53 (dq,
1H, J ˆ 13.0, 3.4 Hz), 1.35 (qd, 1H, J ˆ 12.8, 3.5 Hz), 1.30 ± 1.15 (m, 3H),
1.04 (qd, 1H, J ˆ 12.6, 3.5 Hz), 0.94 (d, 3H, J ˆ 6.5 Hz), 0.83 (d, 3H, J ˆ
6.4 Hz), 0.66 (tt, 1H, J ˆ 10.6, 3.6 Hz); 13C NMR/DEPT (50 MHz, CDCl3):
2-[(1S,2R,3R,4R)-3-[5-(1-Ethoxyethoxy)-5-methylhex-3-ynyl]-2,4-dime-
thylcyclohexyl-2-methyl-[1,3]-dioxolane (19a): A suspension of sodium
hydride (60% dispersion in mineral oil, 0.079 g, 1.967 mmol) in dimethyl-
sulfoxide (2 mL) was stirred for 90 min at 658C. The resulting mixture was
cooled, and treated with 3-(1-ethoxyethoxy)-3-methyl-1-butyne (0.310 g,
1.967 mmol) and after 5 min with a solution of tosylate 18a (0.078 g,
0.197 mmol) in dimethylsulfoxide (0.3 mL) and tetrahydrofuran (0.3 mL).
After stirring for 2 h, the reaction mixture was poured into an ice-cold
saturated solution of ammonium chloride. After extraction with ether, the
organic phase was dried (MgSO4) and concentrated in vacuo. Purification
of the residue by column chromatography and HPLC (pentane/acetone
97:3) afforded alkyne 19a (0.058 g, 77%). Rf ˆ 0.31 (pentane/acetone 97:3);
ˆ
ˆ
d ˆ 149.5 (C ), 110.6 (CH2 ), 60.7 (CH2-O), 53.9 (CH), 47.4 (CH), 37.6
(CH), 36.2 (CH), 35.7 (CH2), 33.2 (CH2), 32.1 (CH2), 20.6 (CH3), 18.7
‡
(CH3), 17.3 (CH3); MS: m/z (%): 196 (4) [M] , 181 (5), 163 (1), 153 (13), 137
(9), 135 (6), 121 (7), 109 (31), 95 (42), 82 (100), 67 (43), 55 (39), 41 (35);
C13H24O (196.33): calcd C 79.6, H 12.3; found C 79.52, H 12.37.
1-[(1R,2R,3S,4R)-3-(2-Hydroxyethyl)-2,4-dimethylcyclohexyl]ethanone
(16a): A stream of ozone was passed through a solution of alkene 15a
(0.050 g, 0.255 mmol) in dichloromethane (1.4 mL) and methanol (1.1 mL)
at ꢀ408C until persistance of a blue color. After passing of nitrogen
through the solution dimethylsulfide (0.112 mL, 1.528 mL) was added
dropwise at ꢀ208C and the resulting mixture was stirred overnight at 258C.
After concentration in vacuo, the residue was dissolved in ether and the
organic phase washed with water and brine. After drying (MgSO4) and
concentration in vacuo purification of the residue by column chromatog-
raphy and HPLC (pentane/acetone 84:16) afforded ketone 16a (45 mg,
89%). M.p. 428C; Rf ˆ 0.29 (pentane/acetone 84:16); [a]2D0 ˆ ꢀ0.3 (c ˆ 1.26,
IR (NaBr): nÄ ˆ 2981, 1456, 1378, 1250, 1158, 1117, 1079, 1040, 976 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ 5.08 (q, 1H, J ˆ 5.1 Hz), 3.95 (q, 1H, J ˆ
6.6 Hz), 3.88 (m, 1H), 3.82 (m, 2H), 3.68 (dq, 1H, J ˆ 9.2, 7.1 Hz), 3.48 (dq,
1H, J ˆ 9.3, 7.1 Hz), 2.10 (t, 2H, J ˆ 8.3 Hz), 1.95 (m, 1H), 1.75 ± 1.00 (m,
8H), 1.47 (s, 3H), 1.40 (s, 3H), 1.31 (d, 3H, J ˆ 5.2 Hz), 1.20 (s, 3H), 1.17 (t,
3H, J ˆ 7.1 Hz), 1.07 (d, 3H, J ˆ 6.1 Hz), 0.89 (d, 3H, J ˆ 6.4 Hz), 0.70 (tt,
1H, J ˆ 10.2, 3.1 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ 112.2 (O-C-
ꢂ
ꢂ
O), 96.2 (O-CH-O), 85.2 (C ), 82.2 (C ), 70.2 (C-O), 65.2 (CH2-O), 63.0
(CH2-O), 60.7 (CH2-O), 51.0 (CH), 49.5 (CH), 36.7 (CH), 35.4 (CH2), 35.0
(CH), 30.7 (CH3), 30.3 (CH3), 28.5 (CH2), 28.4 (CH2), 22.4 (CH3), 20.7
(CH3), 19.2 (CH3), 17.3 (CH3), 15.3 (CH3), 14.3 (CH3); MS: m/z (%): 365
(1), 307 (2), 229 (2), 203 (1), 87 (100), 73 (16).
CHCl3); IR (NaBr): nÄ ˆ 3406, 2924, 1704, 1448, 1362, 1246, 1168, 1042 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ 3.64 (brt, 2H, J ˆ 7 Hz), 2.16 (m, 1H),
2.13 (s, 3H), 1.75 ± 1.00 (m, 9H), 0.94 (d, 3H, J ˆ 6.4 Hz), 0.86 (d, 3H, J ˆ
6.4 Hz), 0.70 (tt, 1H, J ˆ 10.8, 3.7 Hz); 13C NMR/DEPT (50 MHz, CDCl3):
2-[(1S,2R,3S,4R)-3-[5-(1-Ethoxyethoxy)-5-methylhexyl]-2,4-dimethylcy-
clohexyl-2-methyl-[1,3]-dioxolane (20a): 5% Rhodium on aluminum oxide
(0.048 g) was added to a solution of alkyne 19a (0.053 g, 0.139 mmol) in
ethyl acetate (5.3 mL). The suspension was hydrogenated, filtered through
a short pad of silica gel and the filtrate concentrated in vacuo. The residue
was purified by column chromatography and HPLC (pentane/acetone
98:2) to afford 20a (0.040 g, 75%). Rf ˆ 0.31 (pentane/acetone 98:2); IR
(NaBr): nÄ ˆ 2936, 1446, 1378, 1207, 1123, 1087, 1034, 973, 865 cmꢀ1; 1H NMR
(500 MHz, CDCl3): d ˆ 4.87 (q, 1H, J ˆ 5.2 Hz), 3.95 (q, 1H, J ˆ 6.6 Hz),
3.89 (q, 1H, J ˆ 6.8 Hz), 3.83 (m, 2H), 3.53 (dq, 1H, J ˆ 8.8, 7.1 Hz), 3.45
(dq, 1H, J ˆ 8.8, 7.1 Hz), 1.94 (m, 1H), 1.69 (m, 1H), 1.35 ± 1.00 (m, 13H),
1.26 (d, 3H, J ˆ 5.3 Hz), 1.21 (s, 3H), 1.19 (s, 3H), 1.17 (s, 3H), 1.16 (t, 3H,
J ˆ 6.9 Hz), 1.00 (d, 3H, J ˆ 6.0 Hz), 0.86 (d, 3H, J ˆ 6.4 Hz), 0.65 (tt, 1H,
J ˆ 10.0 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ 112.5 (O-C-O), 93.6
(O-CH-O), 75.7 (C-O), 64.3 (CH2-O), 63.1 (CH2-O), 58.7 (CH2-O), 51.1
(CH), 50.1 (CH), 42.2 (CH2), 36.8 (CH), 35.7 (CH2), 35.1 (CH), 28.9 (CH2),
28.7 (CH2), 26.5 (CH3), 26.3 (CH3), 25.0 (CH2), 24.9 (CH2), 22.0 (CH3), 20.8
(CH3), 19.2 (CH3), 17.3 (CH3), 15.5 (CH3); MS: m/z (%): 369 (1), 295 (5),
233 (1), 131 (5), 109 (3), 87 (100), 73 (48), 43 (22); C23H44O4 (384.60): calcd
C 71.8, H 11.5; found C 71.66, H 11.94.
ˆ
d ˆ 213.5 (C O), 60.4 (CH2-O), 59.4 (CH), 46.6 (CH), 36.8 (CH), 35.7
(CH), 35.0 (CH2), 32.4 (CH2), 29.4 (CH2), 29.3 (CH3), 20.4 (CH3), 17.9
‡
(CH3); MS: m/z (%): 198 (5) [M] , 180 (3), 165 (15), 153 (3), 137 (13), 109
(30), 95 (52), 81 (58), 69 (43), 43 (100); C12H22O2 (198.31): calcd C 72.68, H
11.18; found C 72.63, H 11.16.
2-[(1S,2R,3R,6R)-2,6-Dimethyl-3-(2-methyl-[1,3]-dioxolan-2-yl)cyclohex-
yl]ethanol (17a):
A solution of the methylketone 16a (0.330 g,
1.664 mmol), trimethylorthoformate (0.330 mL, 3.013 mmol), and p-tolue-
nesulfonic acid (0.017 g, 0.089 mmol) in 1,2-ethanediol (0.985 mL,
17.654 mmol) was stirred at 258C for 24 h. The reaction mixture was
quenched with an aqueous solution of sodium bicarbonate. After stirring
for a further 5 min the mixture was extracted with diethyl ether. The
combined extracts were concentrated in vacuo. Column chromatography
and HPLC (pentane/acetone 83:47) afforded acetal 17a (0.335 g, 83%).
M.p. 308C; Rf ˆ 0.38 (pentane/acetone 85:15); [a]2D0 ˆ ꢀ2.1 (c ˆ 1.06,
CHCl3); IR (NaBr): nÄ ˆ 3307, 2880, 1591, 1478, 1455, 1379, 1304, 1210,
1
1120, 1078, 1038, 950 cmꢀ1; H NMR (500 MHz, CDCl3): d ˆ 3.97 (q, 1H,
J ˆ 6.5 Hz), 3.90 (dt, 1H, J ˆ 6.8, 7.2 Hz), 3.84 (m, 2H), 3.62 (m, 2H), 1.96
(dq, 1H, J ˆ 12.6, 3.1 Hz), ꢁ1.74 (m, 3H), 1.24 ± 1.14 (m, 5H), 1.22 (s, 3H),
1.07 (d, 3H, J ˆ 6.0 Hz), 1.00 (m, 1H), 0.93 (d, 3H, J ˆ 6.4 Hz), 0.62 (tt, 1H,
J ˆ 10.2, 3.6 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ 112.3 (O-C-O),
64.2 (CH2-O), 63.1 (CH2-O), 60.8 (CH2-O), 51.1 (CH), 47.7 (CH), 38.0
(CH), 36.5 (CH), 35.5 (2 Â CH2), 28.4 (CH2), 20.8 (CH3), 19.3 (CH3), 17.5
(CH3); MS: m/z (%): 227 (1), 165 (1), 135 (1), 99 (2), 87 (100), 67 (3), 43
(19); C14H26O3 (242.36): calcd C 69.4, H 10.8; found C 69.07, H 10.98.
1-[(1R,2R,3S,4R)-3-[5-Hydroxy-5-methylhexyl)-2,4-dimethylcyclohexyl]-
ethanone (21a): A solution of acetal 20a (0.039 g, 0.101 mmol) in acetone
(3.9 mL) containing a trace amount of pyridinium p-toluenesulfonate and a
few drops of water was heated at reflux for 20 h. After cooling, the solution
was concentrated in vacuo, the residue was dissolved in diethyl ether and
washed with a saturated sodium bicarbonate solution and brine. After
drying (MgSO4) and concentration the residue was purified by column
chromatography and HPLC (pentane/acetone 85:15) to afford 21a
(0.025 g, 92%). Rf ˆ 0.45 (pentane/acetone 86:14); [a]2D0 ˆ ‡5.5 (c ˆ 1.05,
2-[(1S,2R,3R,6R)-2,6-Dimethyl-3-(2-methyl-[1,3]-dioxolan-2-yl)cyclohex-
yl]ethyl toluene-4-sulfonate (18a): Triethylamine (0.13 mL, 0.957 mmol), a
solution of p-toluenesulfonyl chloride (0.091 g, 0.479 mmol) in dichloro-
methane (0.5 mL) and a trace amount of 4-dimethylaminopyridine were
added at 08C to a solution of alcohol 17a (0.059 g, 0.239 mmol) in
dichloromethane (1.7 mL). After stirring for 20 h at 258C the reaction
mixture was concentrated to half its volume and filtered. The filtrate was
CHCl3); IR (NaBr): nÄ ˆ 3421, 2931, 1707, 1458, 1376, 1205, 1170, 904 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ 2.17 (m, 1H), 2.12 (s, 3H), 1.74 (m, 2H),
1.61 ± 1.20 (m, 12H), 1.20 (s, 2  3H), 1.20 (s, 3H), 1.04 (qd, 1H, J ˆ 13.2,
3.5 Hz), 0.89 (d, 3H, J ˆ 6.5 Hz), 0.81 (d, 3H, J ˆ 6.4 Hz), 0.71 (tt, 1H, J ˆ
Chem. Eur. J. 2001, 7, No. 2
ꢀ WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0947-6539/01/0702-0529 $ 17.50+.50/0
529

P. De Clercq et al.
FULL PAPER
afford alcohols 23a (99%), 23b (52%), and 23c (41%), respectively. A
suspension of alcohol 23a (5.5 mg, 0.022 mmol) and pyridinium chloro-
chromate on aluminum oxide (0.070 g, 0.327 mmol) in dichloromethane
(0.55 mL) was stirred for 5 h. After filtration and washing of the precipitate
with diethyl ether, the ether phase was concentrated in vacuo and the
residue purified by column chromatography and HPLC (pentane/acetone
9:1) to afford aldehyde 24a (3 mg, 55%). In the same way were obtained
24b (66%) and 24c (55%).
10.7, 3.5 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ 213.7 (C O), 71.0 (C-
O), 59.5 (CH), 48.8 (CH), 43.9 (CH2), 35.6 (CH), 35.1 (CH2), 34.3 (CH),
29.5 (CH2), 29.3 (3 Â CH3), 28.1 (CH2), 25.2 (CH2), 24.4 (CH2), 20.3 (CH3),
17.7 (CH3); MS: m/z (%): 250 (5), 232 (2), 207 (9), 177 (3), 152 (6), 137 (6),
109 (26), 95 (28), 69 (32), 43 (100); C17H32O2 (268.44): calcd C 76.1, H 12.0;
found C 75.18, H 12.31. In a similar procedure compounds 21b and 21c
were obtained.
ˆ
Compound 21b: Rf ˆ 0.40 (pentane/acetone 87:13); [a]2D0 ˆ ‡39.2 (c ˆ 0.76,
CHCl3); IR (NaBr): nÄ ˆ 2933, 1705 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆ
2.12 (m, 1H), 2.12 (s, 3H), 1.93 (m, 1H), 1.68 ± 0.94 (m, 15H), 1.20 (s, 3H),
1.20 (s, 3H), 0.83 (d, 3H, J ˆ 7.2 Hz), 0.78 (d, 3H, J ˆ 6.4 Hz); 13C NMR/
Compound 24a: Rf ˆ 0.27 (pentane/acetone 88:12); IR (NaBr): nÄ ˆ 2930,
1
1718 cmꢀ1; H NMR (500 MHz, CDCl3): d ˆ 9.51 (d, 1H, J ˆ 4.1 Hz), 1.98
(tt, 1H, J ˆ 11.6 Hz), 1.80 ± 0.80 (m, 15H), 1.21 (s, 3H), 1.21 (s, 3H), 0.90 (d,
3H, J ˆ 6.5 Hz), 0.76 (tt, 1H, J ˆ 10.6, 3.4 Hz).
ˆ
ꢂ
ꢂ
DEPT (50 MHz, CDCl3): d ˆ 213.2 (C O), 84.8 (C ), 82.7 (C ), 65.1 (C-
O), 59.1 (CH), 48.2 (CH), 35.4 (CH), 34.8 (CH2), 34.2 (CH), 31.5 (2 Â CH3),
29.3 (CH3, CH2), 27.3 (CH2), 20.2 (CH3), 17.8 (CH3), 14.0 (CH2); MS: m/z
(%): 249 (10), 203 (3), 161 (5), 133 (8), 107 (11), 95 (17), 43 (100).
Compound 24b: Rf ˆ 0.24 (pentane/acetone 9:1); IR (NaBr): nÄ ˆ 2933,
1
1726 cmꢀ1; H NMR (500 MHz, CDCl3): d ˆ 9.51 (d, 1H, J ˆ 4.0 Hz), 1.95
(m, 2H), 1.70 ± 1.05 (m, 15H), 1.20 (s, 3H), 1.20 (s, 3H), 0.89 (d, 3H, J ˆ
6.4 Hz), 0.84 (d, 3H, J ˆ 7.2 Hz); 13C NMR/DEPT (50 MHz, CDCl3): d ˆ
Compound 21c: Rf ˆ 0.36 (pentane/acetone 85:15); [a]2D0 ˆ ꢀ105.2 (c ˆ
ˆ
205.4 (C O), 71.0 (C-O), 57.8 (CH), 44.9 (CH), 44.0 (CH2), 30.1 (CH), 29.5
1.05, CHCl3); IR (NaBr): nÄ ˆ 3431, 2938, 1705, 1464, 1377, 1356, 1197,
1
905 cmꢀ1; H NMR (500 MHz, CDCl3): d ˆ 2.40 (dt, 1H, J ˆ 11.5, 4.1 Hz),
(CH2), 29.3 (2 Â CH3), 28.4 (CH), 27.5 (CH2), 24.7 (CH2), 20.5 (CH2), 18.2
(CH3), 12.2 (CH3); MS: m/z (%): 237 (3), 191 (3), 179 (1), 153 (3), 137 (5),
125 (5), 109 (27), 81 (17), 59 (100), 43 (31).
2.34 (m, 1H), 2.12 (s, 3H), 1.70 (dq, 1H, J ˆ 13.2, 3.5 Hz), 1.65 ± 0.10 (m,
13H), 1.22 (s, 3H), 1.22 (s, 3H), 0.92 (qd, 1H, J ˆ 12.8, 5.2 Hz), 0.85 (d, 3H,
J ˆ 6.4 Hz), 0.62 (d, 3H, J ˆ 7.1 Hz); 13C NMR/DEPT (50 MHz, CDCl3):
Compound 24c: Rf ˆ 0.31 (pentane/acetone 9:1); IR (NaBr): nÄ ˆ 3398,
ˆ
d ˆ 211.8 (C O), 70.8 (C-O), 56.0 (CH), 47.7 (CH), 43.8 (CH2), 35.0 (CH2),
2934, 1723, 1462, 1377, 1159, 908 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ
31.7 (CH), 30.6 (CH), 29.9 (CH2), 29.2 (2 Â CH3), 28.1 (CH3), 27.4 (CH2),
24.7 (CH2), 21.0 (CH2), 20.2 (CH3), 7.7 (CH3); MS: m/z (%): 250 (3), 235
(2), 207 (9), 195 (3), 165 (3), 152 (5), 123 (9), 109 (17), 87 (64), 69 (45), 43
(100).
9.67 (s, 1H), 2.45 (m, 1H, SJ ˆ 28 Hz), 2.29 (dt, 1H, J ˆ 12.6, 3.6 Hz), 1.74
(m, 2H), 1.60 ± 1.05 (m, 12H), 1.22 (s, 3H), 1.22 (s, 3H), 0.97 (qd, 1H, J ˆ
12.8, 3.4 Hz), 0.87 (d, 3H, J ˆ 7.0 Hz), 0.70 (d, 3H, J ˆ 7.1 Hz); MS: m/z
(%): 236 (3), 207 (2), 191 (2), 165 (3), 139 (3), 123 (5), 95 (21), 69 (15), 59
(100).
Electrochemical oxidation of ketones 21a, 21b, and 21c: The electro-
chemical oxidation was performed in an undivided cell equipped with two
platinum electrodes under magnetic stirring at 08C (external cooling). A
solution of methyl ketone 21a (0.090 g, 0.335 mmol) and sodium bromide
(0.069 g, 0.671 mmol) in methanol (3 mL) was brought under a tension of
20 ± 30 V for 45 min. The reaction mixture was concentrated in vacuo and
extracted with diethyl ether. The combined organic phases were washed
with water, dried (MgSO4) and concentrated in vacuo. Column chroma-
tography and HPLC (pentane/acetone 88:12) afforded methyl ester 22a
(0.027 g, 28%). Similarly methyl ketones 21b (0.042 g) and 21c (0.100 g)
led to the corresponding esters 22b (46%) and 22c (76%).
Wittig ± Horner coupling reaction of aldehydes 24a, 24b, and 24c with
phosphine oxide 25: n-Butyllithium (0.041 mL, 2.5m hexanes, 0.104 mmol)
was added dropwise at ꢀ788C to a solution of phosphine oxide 25 (0.060 g,
0.104 mmol) in dry tetrahydrofuran (0.42 mL). After stirring for 20 min a
solution of aldehyde 24a (0.004 g, 0.016 mmol) in tetrahydrofuran
(1.05 mL) was added very slowly to the deep red reaction mixture. After
stirring for 3 h at ꢀ788C, the reaction mixture was warmed slowly to 08C.
After stirring for a few minutes exposed to air (the reaction mixture turned
to yellow), the mixture was directly loaded on a silica gel column (pentane/
ether 7:3). The crude product was dissolved in tetrahydrofuran (1.75 mL)
and treated with tetrabutylammonium fluoride (0.35 mL, 1m THF). After
stirring overnight at 258C, the mixture was concentrated in vacuo and the
residue purified by column chromatography and HPLC (pentane/acetone
7:3) to afford analogue 4a (4.6 mg, 57%). In the same way aldehydes 24b
and 24c were converted into analogues 4b (57%) and 4c (49%),
respectively.
Compound 22a: Rf ˆ 0.31 (pentane/acetone 9:1); [a]2D0 ˆ ꢀ13.9 (c ˆ 0.64,
CHCl3); IR (NaBr): nÄ ˆ 3395, 2932, 1737, 1456, 1380, 1256, 1195, 1141 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d ˆ 3.67 (s, 3H), 2.03 (td, 1H, J ˆ 11.6,
3.1 Hz), 1.81 (dq, 1H, J ˆ 12.9, 3.2 Hz), 1.71 (dq, 1H, J ˆ 13.3, 3.2 Hz),
1.65 ± 0.95 (m, 13H), 1.2 (s, 3H), 1.20 (s, 3H), 0.89 (m, 6H), 0.71 (tt, 1H, J ˆ
10.7 Hz); 13C NMR (50 MHz, CDCl3): d ˆ 176.9, 70.9, 51.8, 51.2, 48.6, 43.8,
36.1, 34.7, 34.0, 29.9, 29.7, 29.1, 28.1, 25.0, 24.2, 20.2, 17.7; MS: m/z (%): 269
(15), 266 (29), 253 (13), 237 (19), 226 (58), 219 (10), 206 (38), 191 (33), 169
(18), 151 (18), 137 (41), 126 (38), 123 (20), 109 (92), 95 (41), 81 (37), 69 (47),
59 (100), 43 (48).
Compound 4a: Rf ˆ 0.29 (pentane/acetone 7:3); IR (NaBr): nÄ ˆ 3395, 2929,
1450, 1378, 1048, 970, 908, 735 cmꢀ1; UV (MeOH): lmax ˆ 209, 250 nm;
1H NMR (500 MHz, CDCl3): d ˆ 6.33 (dd, 1H, J ˆ 15.2, 10.8 Hz), 6.03 (d,
1H, J ˆ 10.7 Hz), 5.52 (dd, 1H, J ˆ 15.3, 8.9 Hz), 5.30 (s, 1H), 5.00 (s, 1H),
4.44 (m, 1H), 4.21 (m, 1H), 2.57 (dd, 1H, J ˆ 13.1, 9.8 Hz), 2.26 (dd, 1H,
J ˆ 13.1, 7.5 Hz), 2.00 ± 0.95 (m, 20H), 1.20 (s, 3H), 1.20 (s, 3H), 0.87 (d, 3H,
J ˆ 6.4 Hz), 0.82 (d, 3H, J ˆ 6.4 Hz), 0.67 (m, 1H); MS: m/z (%): 372 (3),
351 (21), 319 (3), 303 (9), 273 (3), 225 (7), 199 (45), 183 (19), 135 (36), 105
(36), 77 (55), 43 (100).
Compound 22b: Rf ˆ 0.25 (pentane/acetone 9:1); [a]2D0 ˆ ‡14.2 (c ˆ 0.90,
CHCl3); IR (NaBr): nÄ ˆ 3417, 2935, 1738, 1454, 1381, 1257, 1196, 1168, 1144,
1021, 909 cmꢀ1; 1H NMR (500 MHz, CDCl3): d ˆ 3.67 (s, 3H), 2.00 (td, 1H,
J ˆ 11.5, 3.9 Hz), 1.93 (m, 1H), 1.70 ± 0.90 (m, 15H), 1.21 (s, 2  3H), 0.85
(d, 3H, J ˆ 7.1 Hz), 0.82 (d, 3H, J ˆ 6.3 Hz); 13C NMR/DEPT (50 MHz,
ˆ
CDCl3): d ˆ 17ss7.0 (C O), 71.0 (C-O), 52.2 (CH), 51.3 (CH3-O), 45.2
(CH), 44.0 (CH2), 32.7 (CH), 32.5 (CH2), 29.8 (CH2), 29.2 (2 Â CH3), 28.7
(CH), 27.4 (CH2), 24.7 (CH2), 24.2 (CH2), 18.0 (CH3), 12.1 (CH3); MS: m/z
(%): 266 (3), 226 (5), 206 (6), 167 (5), 137 (7), 109 (30), 95 (16), 59 (100).
Compound 4b: Rf ˆ 0.30 (pentane/acetone 7:3); IR (NaBr): nÄ ˆ 3354,
2912 cmꢀ1; UV (MeOH): lmax ˆ 209, 248 nm; 1H NMR (500 MHz, CDCl3):
d ˆ 6.32 (dd, 1H, J ˆ 15.2, 10.9 Hz), 6.03 (d, 1H, J ˆ 10.8 Hz), 5.53 (dd, 1H,
J ˆ 15.2, 9.0 Hz), 5.31 (s, 1H), 4.99 (s, 1H), 4.43 (brs, 1H), 4.21 (brs, 1H),
2.57 (dd, 1H, J ˆ 13.4, 3.6 Hz), 2.26 (dd, 1H, J ˆ 13.0, 7.4 Hz), 2.00 ± 0.90 (m,
21H), 1.21 (s, 3H), 1.21 (s, 3H), 0.81 (d, 3H, J ˆ 7.2 Hz), 0.82 (d, 3H, J ˆ
6.0 Hz); MS: m/z (%): 372 (5), 354 (3), 336 (3), 255 (1), 191 (3), 166 (9), 148
(66), 109 (44), 95 (50), 43 (100).
Compound 22c: Rf ˆ 0.27 (pentane/acetone 9:1); [a]2D0 ˆ ꢀ51.0 (c ˆ 0.82,
CHCl3); IR (NaBr): nÄ ˆ 3413, 2936, 1735, 1458, 1379, 1204, 1133, 1021,
1
908 cmꢀ1; H NMR (500 MHz, CDCl3): d ˆ 3.67 (s, 3H), 2.41 (dt, 1H, J ˆ
11.0, 4.8 Hz), 2.30 (m, 1H, SJ ˆ 28 Hz), 1.75 ± 0.90 (m, 15H), 1.21 (s, 3H),
1.21 (s, 3H), 0.85 (d, 3H, J ˆ 6.4 Hz), 0.69 (d, 3H, J ˆ 7.1 Hz); 13C NMR/
ˆ
DEPT (50 MHz, CDCl3): d ˆ 175.8 (C O), 71.0 (C-O), 51.4 (CH3-O), 48.0
Compound 4c: Rf ˆ 0.25 (pentane/acetone 7:3); IR (NaBr): nÄ ˆ 3339, 2930,
1455, 1380, 1057, 976, 908 cmꢀ1; UV (MeOH): lmax ˆ 207, 247 nm; 1H NMR
(500 MHz, CDCl3): d ˆ 6.34 (dd, 1H, J ˆ 15.3, 10.8 Hz), 6.05 (d, 1H, J ˆ
10.8 Hz), 5.74 (dd, 1H, J ˆ 15.4, 6.7 Hz), 5.31 (brs, 1H), 5.01 (brs, 1H), 4.44
(m, 1H), 4.22 (m, 1H), 2.57 (dd, 1H, J ˆ 13.3, 3.4 Hz), 2.27 (dd, 1H, J ˆ
13.3, 6.9 Hz), 2.14 (m, 1H), 2.00 ± 0.95 (m, 18H), 1.85 (m, 1H), 1.66 (dq, 1H,
J ˆ 13.1, 3.4 Hz), 1.21 (s, 3H), 1.21 (s, 3H), 0.84 (d, 3H, J ˆ 6.4 Hz), 0.66 (d,
3H, J ˆ 7.1 Hz); MS: m/z (%): 372 (5), 354 (2), 314 (1), 278 (1), 255 (1), 223
(1), 207 (3), 166 (12), 148 (50), 109 (51), 91 (48), 43 (100).
(CH), 47.3 (CH), 44.0 (CH2), 35.2 (CH2), 32.1 (CH), 30.5 (CH), 30.0 (CH2),
29.2 (2 Â CH3), 27.4 (CH2), 24.8 (CH2), 21.9 (CH2), 20.2 (CH3), 8.1 (CH3);
MS: m/z (%): 266 (8), 237 (3), 226 (6), 191 (12), 170 (3), 167 (9), 137 (15),
109 (28), 95 (23), 81 (37), 59 (100).
Conversion of esters 22a, 22b, and 22c into the corresponding aldehydes
24a, 24b, and 24c: Esters 22a, 22b, and 22c were reduced with lithium
aluminumhydride in tetrahydrofuran to the corresponding alcohols, as
described for the conversion of ester 13a into primary alcohol 15a, to
530
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Chem. Eur. J. 2001, 7, No. 2

1a,25-Dihydroxyvitamin D3 Analogues
520±532
Conformational analysis and molecular modeling: Conformational analysis
of the side chain of compounds 4a, 4b, and 4c was carried out using the
MacroModel molecular modeling program of Still[44] run on a Digital
VAXstation 4000 ± 90A or SiliconGraphics Octane. Molecular mechanics
calculations were carried out on model compounds in which the A-ring and
diene system up to C6 were replaced by a H atom. Rotations with 608
[8] S. J. Yung, Y. Y. Lee, S. De Vos, E. Elstner, A. W. Norman, J. Green,
M. R. Uskokovic, H. P. Koeffler, Leuk. Res. 1994, 101, 713 ± 718.
[9] L. Binderup, S. Latini, E. Binderup, C. Bretting, M. Calverley, K.
Hansen, Biochem. Pharmacol. 1991, 42, 1569 ± 1575.
[10] a) G. H. Posner, J. K. Lee, Q. Wang, S. Peleg, M. Burke, H. Brem, P.
Dolan, T. W. Kernler, J. Med. Chem. 1998, 41, 3008 ± 3014; b) T. W.
Kernler, P. D. Dolan, S. J. Gange, J. K. Lee, Q. Wang, G. H. Posner,
Carcinogenesis 2000, 21, 1341 ± 1345.
ꢀ
increments were applied to the rotatable C C bonds of the side chain,
while the 25-OH was rotated with increments of 1208. The starting
conformations thus generated were minimized using the MM2* force field
implementation of MacroModel and the conformations within 20 kJmolꢀ1
of the minimum energy form were retained. Using a PC computer program
all conformations of each compound were then overlaid using C13 as
common origin (x, y, z ˆ 0), C14 was positioned in the y,z plane (x ˆ 0) and
the 18-position was made to coincide with the positive y axis (x, z ˆ 0). A
line drawing was generated of the minimum energy conformation and the
position of O25 in each of the local energy minima within the given energy
window was represented by a ball to obtain the volume maps shown in
Figures 3, 4, and 5.
[11] a) R. Bouillon, P. De Clercq, P. Pirson, M. Vandewalle, Novel
Structural Analogues of Vitamin D. Patent PCT/EP 93.202037.3,
1993 [Chem. Abstr. 1996, 122, 314933m]; b) K. Sabbe, C. D'Halleweyn,
P. De Clercq, M. Vandewalle, R. Bouillon, A. Verstuyf, Bioorg. Med.
Chem. Lett. 1996, 6, 1697 ± 1702; c) G.-D. Zhu, Y. Chen, X. Zhou, M.
Vandewalle, P. J. De Clercq, R. Bouillon, A. Verstuyf, Bioorg. Med.
Chem. Lett. 1996, 6, 1703 ± 1708; d) Y. J. Chen, P. De Clercq, M.
Vandewalle, Tetrahedron Lett. 1996, 37, 9361 ± 9364; e) Y. Wu, C.
DꢁHalleweyn, D. Van Haver, P. De Clercq, M. Vandewalle, Bioorg.
Med. Chem. Lett. 1997, 7, 923 ± 928; f) B. Linclau, P. De Clercq, M.
Vandewalle, Bioorg. Med. Chem. Lett. 1997, 7, 1461 ± 1468; g) A.
Verstuyf, L. Verlinden, S. Ling, Y. Wu, C. DꢁHalleweyn, D. Van Haver,
G.-D. Zhu, D. Zhu, Y. Chen, X. Zhou, H. Van Baelen, P. De Clercq,
M. Vandewalle, R. Bouillon, J. Bone Miner. Res. 1998, 13, 549 ± 558;
h) X. Zhou, G.-D. Zhu, D. Van Haver, M. Vandewalle, P. J. De Clercq,
A. Verstuyf, R. Bouillon, J. Med. Chem. 1999, 42, 3539 ± 3556; i) A.
Verstuyf, L. Verlinden, E. Van Etten, L. Shi, Y. Wu, C. DꢁHalleweyn,
D. Van Haver, G.-D. Zhu, Y.-J. Chen, X. Zhou, M. R. Haussler, P.
De Clercq, M; Vandewalle, H. Van Baelen, C. Mathieu, R. Bouillon, J.
Bone Miner. Res. 2000, 15, 237 ± 252.
Biological evaluation: Compound 2 (MC 1288) was a kind gift of L.
Binderup (Leo Pharmaceuticals, Ballerup, Denmark).
Binding studies: The affinity of the 6D-analogues of 1,25(OH)2D3 to the
vitamin D receptor was evaluated in their ability to compete with
[3H]1,25(OH)2D3 for binding to high speed supernatant from intestinal
mucosa homogenates obtained from normal pigs as described previous-
ly.[11g] The relative affinity of the analogues was calculated from their
concentration needed to displace 50% of [3H]1,25(OH)2D3 from its
receptor compared with the activity of 1,25(OH)2D3.
MCF-7 proliferation assay: The human breast carcinoma (MCF-7) cell line
was obtained from the American Tissue Culture Company (Rockville,
MD). The antiproliferative activity of 1,25(OH)2D3 or 6D analogues on
MCF-7 cells was assessed by evaluating [3H]thymidine incorporation. Cells
were seeded in 96-well plates (7500 cells per well) and 1 mCi [methyl-
3H]thymidine (ICN Biomedicals, Costa Mesa, CA) was added 72 h after the
initiation of treatment. Cells were semi-automatically harvested after an
additional 6 h of incubation on filter plates only retaining incorporated
thymidine (GF/C Filter and Filtermate Universal Harvester, Packard
Instrument, Meriden, CT). Counting was performed using a microplate
scintillation counter (Topcount, Packard).
[12] Whereas the VDR affinity of 2 is often higher than that of 1
(chicken,[9] bovine thymus), in our model (pig) the opposite is found;
see: a) F. J. Dilworth, M. J. Calverley, H. L. Makin, G. Jones, Biochem.
Pharmacol. 1994, 47, 987 ± 993; b) K. Yamamoto, H. Ooizumi, K.
Umesono, A. Verstuyf, R. Bouillon, H. F. DeLuca, T. Shinki, T. Suda,
S. Yamada, Bioorg. Med. Chem. Lett. 1999, 9, 1041 ± 1046.
[13] a) R. A. Scott, H. A. Scheraga, J. Chem. Phys. 1966, 44, 3054; b) A.
Abe, R. L. Jernigan, P. J. Flory, J. Am. Chem. Soc. 1966, 88, 631 ± 639;
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7919; f) H. Goto, E. Osawa, M. Yamato, Tetrahedron 1993, 49, 387 ±
396.
Differentiation of HL-60 cells: The human promyelocytic leukemia cell
line (HL-60) was obtained from the American Tissue Culture Company
(Rockville, MD). HL-60 cells were seeded at 4 Â 104 cells mLꢀ1 in 25 cm2
Falcon tissue chambers using RPMI 1640 medium supplemented with 20%
[14] For a seminal example of the design of a monoconformational
polyether, see: a) T. Iimori, S. D. Erickson, A. L. Rheingold, W. C.
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fetal calf serum (Sera-Lab, W. Sussex, UK) and gentamycin (50 mg mLꢀ1
;
Gibco, Roskilde, Denmark). 1,25(OH)2D3, analogues, or vehicle were
added the day after plating. After 4 d of culture, differentiation was
measured by using the NBT reduction assay as described previously.[45]
[15] For an authorative description of the concept, see: R. W. Hoffman, M.
Stahl, M. Schopfer, G. Frenking, Chem. Eur. J. 1998, 4, 459 ± 566.
‡
[16] In the g -arrangement shown C22 suffers two gauche interactions;
In vivo studies: NMRI mice were obtained from the Proefdierencentrum of
Leuven (Belgium) and fed on a vitamin D-replete diet (0.2% calcium, 1%
phosphate, 2000 U vitamin D kgꢀ1; Hope Farms, Woerden, The Nether-
lands). The hypercalcemic effect of the 6D analogues was tested in NMRI
mice by daily subcutaneous injection of 1,25(OH)2D3, its analogues or the
solvent during seven consecutive days, using serum calcium concentration
as parameter.
hence there may be a tendency to populate conformers with an
[15]
ꢀ
eclipsed arrangement at C17 C20;
J. E. Anderson, A. I. Ijek, J.
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[19] The calculated equilibrium composition on the basis of steric energies
obtained using the MacroModel molecular modeling program of
Still[44] equals 72:28 in favor of 6a.
[20] F. Camps, J. Coll, J. Guitart, Tetrahedron 1986, 42, 4603 ± 4609.
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[22] Relative differences in steric energies obtained for the global
minimum energy conformations of 7a, 7b, 7c, and 7d equal 0.0, 9.7,
9.9, and 5.8 kJmolꢀ1 [44]
.
[23] For the application of a similar strategy in a related project, see: S.
È
Gabriels, D. Van Haver, M. Vandewalle, P. De Clercq, D. Viterbo, Eur.
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Chem. Eur. J. 2001, 7, No. 2
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0947-6539/01/0702-0531 $ 17.50+.50/0
531

P. De Clercq et al.
FULL PAPER
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Received: June 7, 2000 [F2529]
532
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Chem. Eur. J. 2001, 7, No. 2