Synthesis of 9-alkylated calcitriol and two 1α,25-dihydroxy-9- methylene-10,19-dihydrovitamin D3 analogues with a non-natural triene system by thermal sigmatropic rearrangements

Article abstract of DOI:10.1021/jo302513e

1α,25-(OH)₂-9α-Methylvitamin D₃ (4), the first known analogue of the natural hormone 1α,25-(OH)₂D ₃ (3) with an alkyl substituent at C-9, and two 1α,25-(OH) ₂-9-methylene-10,19-dihydrovitamin D₃ analogues (7 and 8) with an unprecedented non-natural triene system were synthesized by thermal isomerization of 1α,25-(OH)₂-9-methylprevitamin D₃ (6). Three alternative approaches (Sonogashira, Stille, or stereoselective dehydration of a tertiary propargyl alcohol) have been successfully used to construct the dienyne precursors of previtamin 6 possessing two methyl groups capable of participating in the [1,7]-sigmatropic hydrogen shift.

Full text of DOI:10.1021/jo302513e

Article
pubs.acs.org/joc
Synthesis of 9‑Alkylated Calcitriol and Two 1α,25-Dihydroxy-9-
methylene-10,19-dihydrovitamin D3 Analogues with a Non-natural
Triene System by Thermal Sigmatropic Rearrangements
Urszula Kulesza,† Rita Sigueiro,‡ Antonio Mourino,*,‡ and Rafal R. Sicinski*,†
̈
̃
†Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
‡Departamento de Quimica Organica y Unidad Asociada al CSIC, Universidad de Santiago de Compostela, 15782 Santiago de
Compostela, Spain
S
* Supporting Information
ABSTRACT: 1α,25-(OH)2-9α-Methylvitamin D3 (4), the first known analogue of the natural hormone 1α,25-(OH)2D3 (3)
with an alkyl substituent at C-9, and two 1α,25-(OH)2-9-methylene-10,19-dihydrovitamin D3 analogues (7 and 8) with an
unprecedented non-natural triene system were synthesized by thermal isomerization of 1α,25-(OH)2-9-methylprevitamin D3 (6).
Three alternative approaches (Sonogashira, Stille, or stereoselective dehydration of a tertiary propargyl alcohol) have been
successfully used to construct the dienyne precursors of previtamin 6 possessing two methyl groups capable of participating in
the [1,7]-sigmatropic hydrogen shift.
(10),6,8(14)-trienes labeled with deuterium at the C-15
position.
INTRODUCTION
■
1α,25-Dihydroxyvitamin D3 [calcitriol, 1α,25-(OH)2D3 (3);
Figure 1] is the most potent vitamin D3 (2) metabolite, a
hormonal form responsible for calcium and phosphorus
homeostasis.1 Its genomic actions are mediated through the
vitamin D receptor (VDR),2,3 a member of the nuclear receptor
superfamily,4 whose established presence in more than 30
tissues5 indicates that 1α,25-(OH)2D3 may also be involved in a
broad array of physiological processes not yet known. A
metabolic precursor of 2 is isomeric previtamin D3 (1), which
in turn is produced in the skin by UV irradiation of 7-
dehydrocholesterol.6 For a conversion of previtamin D3 to
vitamin D3, the temperature of the human body is sufficient; at
room temperature an equilibrium 1 ⇆ 2 (8:92) is reached after
few days (t1/2 = 70 h).7 This thermally induced process of
crucial biological importance represents an elegant example of
[1,7]-sigmatropic hydrogen shift defined by Woodward and
Hoffmann.8 Kinetic and thermodynamic aspects of such
pericyclic process have attracted chemists’ attention for more
than a half of century.7,9 The intramolecular nature of this
hydrogen shift was established long ago10 and supported by
studies involving 19-substituted vitamin D analogues.11
Antarafacial stereochemistry of the thermal [1,7]-hydrogen
shift, as predicted by Woodward−Hoffmann rules,8,12 was
demonstrated by the Okamura group in their studies on cis-
isotachysterol analogues.13 To achieve this goal, the authors
designed the conjugated 1-hydroxy-9,10-seco-cholesta-5-
The discovery of calcitriol and its role in living organisms,
extending far beyond the classical regulation of calcium and
phosphorus levels, stimulated studies directed to the synthesis
of its analogues with modified biological activity.14 Among
more than 3000 such compounds synthesized to date, only very
few of them were characterized by substitution at C-9. Thus, in
1991, Mourino and co-workers reported the preparation of the
̃
9α-hydroxy analogue of calcitriol with a different configuration
of the intercyclic diene moiety,15 and in the more recent
Japanese patent, several 9α-alkylated 19-norvitamin D com-
pounds were described.16 Therefore, as an extension of the
structure−activity studies carried out in our laboratories, we
designed new homologues of calcitriol methylated at C-9 (4
and 5). Their precursor, previtamin D compound 6, would
possess a conjugated hexatriene moiety, symmetrically
substituted with two methyl groups at the terminal carbons
C-9 and C-10. Examination of the thermal rearrangement of
such an unsaturated system seemed to be an attractive endeavor
because two competitive [1,7]-hydrogen shifts were envisioned,
occurring from both methyl substituents located at the terminal
carbons of the hexatriene moiety. The first process, involving
methyl located at C-10, would result in the formation of the
Received: November 16, 2012
Published: January 14, 2013
© 2013 American Chemical Society
1444
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450

The Journal of Organic Chemistry
Article
Figure 1. Structures of the 1α,25-(OH)2D3, its metabolic precursors, the synthesized vitamin D3 analogues, and their synthetic precursors.
Scheme 1. Synthesis of Dienyne 15 via Sonogashira Coupling
target 9-methyl calcitriol analogues 4 and 5 whereas the
alternative sigmatropic shift of hydrogen from the methyl
attached to C-9 would produce 9-methylene-10,19-dihydrovi-
tamins 7 and 8. These latter compounds would be
characterized by a unique triene system.
presence of DMPU, followed by addition of MeI.16 The
stereochemistry of the methylated ketone 11 was easily
determined by analysis of its 1H NMR spectrum and molecular
modeling (PCModel, v9.0, Serena Software). Spin decoupling
experiment performed with the multiplet derived from the
methine proton at C-9 (δ 2.43) allowed for measuring vicinal
coupling constants (6.3 and 2.1 Hz) indicating its equatorial
disposition (the calculated values for 11: J9β,11β = 6.8 Hz, J9β,11α
= 1.2 Hz) and, consequently, axial α-orientation of the
introduced methyl group.
RESULTS AND DISCUSSION
■
We have chosen two known compounds, namely the protected
25-hydroxy Grundmann ketone 917and the enyne 10,18 as
suitable substrates for the synthesis of the analogues 4, 5, 7, and
8. Since a strategy of our synthesis involved Sonogashira
coupling, we first transformed the starting hydrindanone 9 into
vinyl iodide 13 (Scheme 1). α-Methylation of 9 was achieved in
almost quantitative yield by its treatment with LDA in the
The following conversion of the ketone 11 into vinyl iodide
was performed by application of the Barton’s procedure.19 The
intermediate hydrazones were reacted with iodine, and the
formed isomeric vinyl iodides, after hydroxyl deprotection, were
1445
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450

The Journal of Organic Chemistry
Article
Scheme 2. Synthesis of 1α,25-Dihydroxy-9-methylprevitamin D3 (6) via Stille Coupling
Scheme 3. Synthesis of 1α,25-Dihydroxy-9-methylprevitamin D3 (6) via Acetylide Addition to Hydrindanone and Its Thermal
Isomerization Process
effectively separated by reversed-phase HPLC. The desired
prevailing isomer 13 was then coupled to A-ring synthon 10 in
the presence of bis[triphenylphosphine]palladium(II) chloride-
copper(I) iodide catalyst and diethylamine.20 The dienyne 15
was obtained in excellent yield using a 5-fold molar excess of
the starting enyne 10.21 We also explored an alternative
approach22 to dienyne 15 to avoid the use of an excess of A-
ring fragment. Thus, treatment of tributyltin derivative 16
(Scheme 2), prepared from enyne 10, with an equimolar
amount of the iodide 13 provided dienyne 15 in good yield.
The resulting dienyne 15 was subsequently semihydrogenated
in the presence of Lindlar catalyst and quinoline to afford the
triene 17 which was deprotected to previtamin D 6. The UV
spectrum of the latter compound shows an absorption
maximum at 248 nm, that differs considerably from those of
previtamins unsubstituted at C-9 (λmax 261 nm). The observed
blue shift in the UV spectrum can be attributed, as in the case
of 6-methylprevitamin D3 described by Sheves and Mazur,23 to
the increased deviation from planarity of the hexatriene
chromophore, caused by steric interaction of its additional
alkyl substituent. Interestingly, the UV spectrum of 6 is similar
to that reported for the 6-methylated analogue (λmax 247 nm).23
In an attempt to improve the yield of previtamin D
compound 6, a new synthetic method was developed. Thus,
1446
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450

The Journal of Organic Chemistry
Article
the methylated Grundmann ketone 11 was treated with an
anion of the enyne 10 to afford the enynol 18 (Scheme 3),
which underwent syn-elimination upon reaction with the
Burgess reagent24 to provide the desired dienyne 19 in high
yield. The stereochemical outcome of this process confirmed an
attack of an acetylide anion on ketone 11 from the α-side
despite the presence of 9α-methyl substituent. The observed
chemical shift of the angular methyl group at C-13 (δ 0.93) in
18 is similar to that observed in the analogous 8β-hydroxy-des-
A,B-steroid.25 Compound 19 was then successfully converted
to the desired previtamin D 6 using the method described
above.
With the 9-methylprevitamin D compound 6 in hand, we
proceeded to study its thermal sigmatropic behavior. The
thermal isomerization was carried out in refluxing isooctane,
and its progress was carefully monitored by HPLC. The
equilibrium state was reached after approximately 14 h when no
further significant change in the product composition was
observed. HPLC separation provided the starting previtamin 6
(40%) and three isomeric products 4, 7, and 8 in 6%, 28%, and
15% yield, respectively. The C(9)-epimer of 4, another
theoretically possible reaction product, was not found in the
equilibrium mixture. This observation was not surprising,
because the presence of equatorial 9β-methyl substituent in the
isomeric compound 5 would result in its strong A(1,3)-strain
interaction with the vinyl hydrogen at C-6.26,27 HPLC
monitoring of the thermal reaction indicated that isomer 7
formed faster than 8, and after prolonged heating, this
compound was still the prevailing product. Thus, hydrogen
from the 9-methyl group migrates to C-10 preferentially in a syn
manner to the 1α-hydroxyl group. A similar syn-directing effect
was observed by Okamura during the studies on thermal
rearrangement of isotachysterol derivatives.13 Not unexpect-
edly, the UV spectra of compounds 7 and 8, possessing “the
rotated” triene chromophore, were almost identical to the
respective spectra of the vitamin D hormone 3 and its currently
synthesized 9α-methylated homologue 4.
the synthesis involves the generation of the natural and non-
natural vitamin D triene moieties from a common previtamin D
compound by thermal sigmatropic [1,7]-H shifts. The required
previtamin D 6 was synthesized through three alternative
approaches: Sonogashira, Stille, and stereoselective dehydration
of a propargylic alcohol by Burgess reagent.
EXPERIMENTAL SECTION
■
(1R,3aR,5R,7aR)-1-[(R)-1′,5′-Dimethyl-5′-[(triethylsilyl)oxy]-
hexyl]-5,7a-dimethyloctahydroinden-4-one (11). A solution of
ketone 9 (400 mg, 1.01 mmol) in anhydrous THF (5 mL) was added
dropwise to a solution of LDA (2.0 M in THF/heptane/ethylbenzene;
1.01 mL, 2.02 mmol) in THF (3 mL) under argon at −78 °C,
followed by addition of DMPU (95 μL, 0.8 μmol). The solution was
warmed to −30 °C and stirred at this temperature for 1 h. Then the
resulting mixture was cooled to −78 °C, and methyl iodide (4.05
mmol, 0.25 mL) was added. The reaction mixture was allowed to reach
−30 °C over 1 h, stirred at this temperature for 3 h, and then
quenched by addition of water. The product was extracted with ether,
and the combined organic extracts were washed with brine, dried
(MgSO4), and evaporated in rotary evaporator. The remaining oily
residue was purified by flash chromatography. Elution with hexane/
Et2O (98:2) gave the methylated ketone 11 (396 mg, 96%) as a
colorless oil: [α]24 −29.3 (c 2.0, CHCl3); 1H NMR (500 MHz,
D
CDCl3) δ 0.562 (6H, br q, J = 8.0 Hz, 3 × SiCH2), 0.650 (3H, s, CH3-
7a), ca. 0.93 (3H, d, J ≈ 6.5 Hz, CH3-1′), 0.945 (9H, t, J = 8.0 Hz, 3 ×
SiCH2CH3), 1.183 (3H, d, J = 7.2 Hz, CH3-5), 1.189 (6H, s, CH3-5′
and H3-6′), 2.43 (1H, m, H-5), 2.66 (1H, dd, J = 11.0, 7.6 Hz, H-3a);
13C NMR (125 MHz, CDCl3) δ 7.0, 7.3, 13.3, 18.7, 18.8, 19.2, 20.9,
27.8, 30.0, 30.2, 30.5, 35.3, 35.7, 36.4, 44.0, 45.6, 49.8, 56.9, 57.5, 73.6,
216.3; HRMS (ESI) exact mass calcd for C25H48O2SiNa (M + Na)+
431.3321, found 431.3346.
(R)-6-[(1′ R, 3a′R, 7a′R)-4-Iodo-5′, 7a′-dimethyl-
2′,3′,3a′,6′,7′,7a′-hexahydro-1H-inden-1-yl)-2-methylheptan-
2-ol (13) and (R)-6-[(1′R,5′R,7a′R)-4-Iodo-5′,7a′-dimethyl-
2′,3′,5′,6′,7′,7a′-hexahydro-1H-inden-1-yl)-2-methylheptan-2-
ol (14). A solution of ketone 11 (300 mg, 0.73 mmol) in EtOH (1
mL) was added to a solution of hydrazine (80% in water; 40 μL, 0.81
mmol) in EtOH (0.2 mL). The reaction mixture was refluxed for 2 h,
and then the solvent was evaporated under vacuum. The residue was
dissolved in ether, and the resulting solution was washed with water,
dried (MgSO4), and concentrated. The oily product 12 was dried in
vacuo for 2 h and then dissolved in toluene (5 mL). To this solution
was added tetramethylguanidine (0.32 mL, 2.57 mmol), followed by a
solution of iodine (196 mg, 1.54 mmol) in toluene (7 mL). The
mixture was stirred at room temperature for 2 h and then quenched by
dropwise addition of 5% aq HCl, poured into saturated Na2S2O3
solution, and extracted with ether. The combined organic extracts were
dried (MgSO4) and concentrated to give a residue which was applied
on a silica Sep-Pak (5 g) and eluted with hexane/Et2O (95:5). The
crude product (200 mg) was dissolved in anhydrous THF (10 mL),
and TBAF (1.0 M in THF; 0.70 mL, 0.70 mmol) was added at room
temperature. The reaction mixture was stirred overnight and quenched
by addition of brine, extracted with ether, dried (MgSO4), and
concentrated. The residue was applied on a silica Sep-Pak (5 g) and
eluted with hexane/Et2O (98:2) to give a mixture of isomeric iodides
13 and 14. Final separation of the obtained mixture was achieved by
HPLC (9.4 × 250 mm Agilent Eclipse XDB-C18 column, 4 mL/min)
using a methanol/water (93:7) solvent system. The vinyl iodide 14
was collected at Rv 51 mL (68 mg, 23% after three steps) and the
The structures of the synthesized compounds 4, 7, and 8
1
were tentatively assigned on the basis of H NMR data. In the
case of vitamin 4, multiplicity of the H-9 signal at 3.07 ppm
(approx quintet, J ≈ 7 Hz) and strong NOE between this
proton and vinylic H-6 (δ 6.38) confirmed its equatorial β-
orientation and, consequently, the presence of the 9α-methyl
group. The proton coupling network in isomeric products 7
and 8 supported the expected preponderance of A-ring chair
conformers in which 10-methyl substituents must be axially
oriented in order to avoid strong allylic interactions.26,27
Therefore, an axial disposition of H-4β in 7 (δ 2.31), and H-1β
in 8 (δ 4.05), deduced from their vicinal coupling constants,
was sufficient to establish configurations at C-10 in these
epimeric compounds; comparison with the spectra of related
10,19-dihydrovitamins28 also supported this assignment.
The biological evaluation of the new synthesized compounds
is underway. The results of preliminary in vitro testing reveal
that a presence of the 9α-methyl group results in significantly
reduced VDR binding activity and HL-60 differentiation
potency of homologue 4 in comparison to 1α,25-(OH)2D3.
isomeric iodide 13 at Rv 56 mL (89 mg, 30% after three steps).
1
13: [α]24 −5.2 (c 2.0, CHCl3); H NMR (200 MHz, CDCl3) δ
D
0.76 (3H, s, CH3-7a′), 0.93 (3H, d, J = 6.4 Hz, H3-7), 1.22 (6H, s, H3-
1 and CH3-2), 1.81 (3H, d, J = 2.4 Hz, CH3-5′), 2.22 (1H, m), 2.46
(1H, m); 13C NMR (50 MHz, CDCl3) δ 11.3, 18.8, 20.8, 27.1, 28.2,
29.2, 29.4, 31.3, 32.3, 36.0, 36.3, 36.5, 44.3, 44.4, 55.5, 56.9, 71.1,
100.6, 137.2; HRMS (ESI) exact mass calcd for C19H33OINa (M +
Na)+ 427.1474, found 427.1452.
CONCLUSIONS
■
We designed and synthesized the first 9-alkylated derivative of
calcitriol (4) and two 1α,25-dihydroxy-9-methylene-10,19-
dihydrovitamin D3 analogues (7 and 8) possessing an
unprecedented non-natural triene system. A key feature of
1447
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450

The Journal of Organic Chemistry
Article
1
14: [α]24 +42.3 (c 1.7, CHCl3); H NMR (200 MHz, CDCl3) δ
acetate, dried (MgSO4), and concentrated. The residue was purified by
HPLC (10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min)
using a hexane/2-propanol (8:2) solvent system. The previtamin D
compound 6 was collected at Rv 50 mL (28 mg, 83%): [α]24D +3.2 (c
0.9, CHCl3); 1H NMR (200 MHz, CDCl3) δ 0.71 (3H, s, H3-18), 0.95
(3H, d, J = 6.6 Hz, H3-21), 1.22 (6H, s, H3-26 and H3-27), 1.44 and
1.77 (3H and 3H, each br s, CH3-9 and H3-19), 4.02 (1H, br m, H-
3α), 4.22 (1H, narr m, H-1β), 5.80 and 6.05 (1H and 1H, each d, J =
12.2 Hz, H-7 and H-6); 13C NMR (50 MHz, CDCl3) δ 11.5, 17.0,
18.7, 19.8, 20.8, 24.1, 28.8, 29.2, 29.4, 31.1, 36.1, 36.4, 36.6, 38.0, 40.6,
42.3, 44.4, 51.8, 54.4, 64.3, 70.9, 71.1, 128.6, 129.2, 130.1, 130.8, 130.9,
131.7; UV (EtOH) λmax = 248 nm (ε =10500); HRMS (ESI) exact
mass calcd for C28H46O3Na (M + Na)+ 453.3345, found 453.3344.
1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9α-methyl-25-
[(triethylsilyl)oxy]-9,10-secocholest-5(10)-en-6-yn-8β-ol (18). n-
Butyllithium (1.56 M in hexane; 556 μL, 0.87 mmol) was added
dropwise to a stirred solution of enyne 10 (300 mg, 0.79 mmol) in
anhydrous THF (4 mL) at −78 °C. After 5 min, CeCl3 (214 mg, 0.87
mmol) was added, and the resulting suspension was stirred at −78 °C
for 30 min. Then a solution of ketone 11 (290 mg, 0.71 mmol) in
THF (4 mL) was added, and the mixture was stirred for 15 min and
warmed to room temperature. The reaction was quenched after 1 h by
addition of saturated solution of NH4Cl. The resulting mixture was
extracted with Et2O, and the combined organic extracts were washed
with brine, dried (MgSO4), and concentrated. The residue was purified
by flash chromatography on silica gel. Elution with hexane/Et2O
(98:2) gave enynol 18 (473 mg, 76%) as a colorless oil: [α]24D −33.8
(c 2.0, CHCl3); 1H NMR (250 MHz, CDCl3) δ 0.07 and 0.10 (6H and
6H, each s, 4 × SiCH3), 0.57 (6H, q, J = 7.1 Hz, 3 × SiCH2), 0.89 and
0.90 (9H and 9H, each s, 2 × Si-t-Bu), ca. 0.90 (3H, d, J ≈ 6.5 Hz, H3-
21), 0.93 (3H, s, H3-18), 0.95 (9H, t, J = 7.1 Hz, 3 × SiCH2CH3), 1.15
(3H, d, J = 7.4 Hz, CH3-9α), 1.19 (6H, s, H3-26 and H3-27), 1.89 (3H,
br s, H3-19), 2.38 (1H, dd, J = 16.1, 5.0 Hz), 4.08 (1H, br m, H-3α),
4.18 (1H, narr m, H-1β); 13C NMR (62 MHz, CDCl3) δ −4.8, −4.7,
−4.6, −4.3, 6.9, 7.2, 13.3, 17.6, 18.1, 18.2, 18.4, 18.8, 20.8, 21.1, 24.7,
25.9, 26.0, 26.8, 29.9, 30.1, 34.5, 35.4, 36.2, 39.5, 39.8, 41.3, 43.0, 45.6,
50.7, 57.1, 64.2, 70.0, 73.5, 74.2, 84.6, 95.5, 115.0, 141.1; HRMS (ESI)
exact mass calcd for C46H88O4Si3Na (M + Na)+ 811.5888, found
811.5866.
D
0.93 (3H, s, CH3-7a′), ca. 0.95 (3H, d, J ≈ 6 Hz, H3-7), 1.16 (3H, d, J
= 7.0 Hz, CH3-5′), 1.22 (6H, s, H3-1 and CH3-2); 13C NMR (50 MHz,
CDCl3) δ 18.3, 18.9, 20.8, 21.8, 26.4, 28.0, 29.4, 29.6, 32.6, 35.1, 35.9,
36.1, 40.7, 44.5, 47.9, 57.0, 71.2, 103.9, 153.9; HRMS (ESI) exact mass
calcd for C19H33OINa (M + Na)+ 427.1474, found 427.1458.
1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-25-hydroxy-9-meth-
yl-9,10-secocholesta-5(10),8-dien-6-yne (15). (a) CuI (6 mg,
0.03 mmol) and Pd(PPh3)2Cl2 (6 mg, 0.01 mmol) were added to a
solution of the iodide 13 (63 mg, 0.16 mmol) and enyne 10 (237 mg,
0.62 mmol) in dry Et2NH (7 mL) at 0 °C. The mixture (protected
from light) was stirred at 0 °C for 1 h and then at room temperature
for 3 h. A saturated solution of NH4Cl was added, and the mixture was
extracted with ether. The combined organic extracts were dried
(MgSO4) and concentrated to give an oily residue which was purified
by flash chromatography on silica gel. Elution with hexane/Et2O
(95:5) afforded dienyne 15 (94 mg, 92%) as a colorless oil: [α]24
−24.8 (c 1.0, CHCl3); H NMR (200 MHz, CDCl3) δ 0.07 and 0.10
D
1
(6H and 6H, each s, 4 × SiCH3), 0.66 (3H, s, H3-18), 0.89 and 0.90
(9H and 9H, each s, 2 × Si-t-Bu), 0.95 (3H, d, J = 7.4 Hz, H3-21), 1.22
(6H, s, H3-26 and H3-27), 1.87 and 1.92 (3H and 3H, each br s, H3-19
and CH3-9), 4.11 (1H, br m, H-3α), 4.20 (1H, narr m, H-1β); 13C
NMR (50 MHz, CDCl3) δ −4.8, −4.7, −4.6, −4.3, 11.2, 18.0, 18.2,
18.7, 19.2, 20.8, 21.1, 24.8, 25.8, 25.9, 28.5, 29.2, 29.4, 30.8, 36.2, 36.3,
36.4, 40.0, 41.4, 42.1, 44.4, 50.6, 54.7, 64.3, 70.1, 71.1, 91.7, 92.4,
115.9, 116.7, 139.5, 140.6; HRMS (ESI) exact mass calcd for
C40H72O3Si2Na (M + Na)+ 679.4918, found 679.4899.
(b) n-Hexyllithium (2.3 M in hexane; 0.34 mL, 0.79 mmol) was
added slowly to a stirred solution of enyne 10 (200 mg, 0.52 mmol) in
THF (4 mL) at −78 °C. After 1 h, freshly distilled tri-n-butyltin
chloride (0.24 mL, 0.84 mmol) was added. The mixture was stirred at
−78 °C for 15 min and then at room temperature for 2 h. The reaction
was quenched by addition of water, diluted with ether, and washed
with saturated NaHCO3 solution. The organic phase was dried
(MgSO4), concentrated, and dried in vacuum. The crude product 16
(260 mg, 0.39 mmol) was dissolved in the solution of the iodide 13
(157 mg, 0.39 mmol) in THF (8 mL). Pd(PPh3)4 (8 mg, 0.01 mmol)
and LiCl (127 mg, 2.99 mmol, dried in vacuum) were added, and the
mixture was refluxed in the dark for 3 h, poured into water, and
extracted with ether. The organic extracts were dried (MgSO4) and
concentrated. The residue was purified by flash chromatography on
silica gel. Elution with hexane/ethyl acetate (98:2) gave the dienyne 15
(211 mg, 83%) as a colorless oil.
1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-9-methyl-25-[(tri-
ethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien-6-yne (19). A
solution of alcohol 18 (100 mg, 0.127 mmol) in toluene (8 mL)
was added dropwise to a solution of methyl N-(triethylammonium-
sulfonyl)carbamate (36 mg, 0.152 mmol) in anhydrous toluene (5
mL). Next, the reaction mixture was heated to 50 °C and stirred at this
temperature for 30 min. Then the mixture was poured into brine and
extracted with ether. The combined organic extracts were dried
(MgSO4) and evaporated to give a residue which was purified by flash
1α-[(tert-Butyldimethylsilyl)oxy]-25-hydroxy-9-methylprevi-
tamin D3 tert-Butyldimethylsilyl Ether (17). Lindlar catalyst (70
mg) and a 0.5% (v/v) solution of quinoline in hexane (0.12 mL) were
added to a solution of dienyne 15 (63 mg, 0.096 mmol) in hexane (10
mL). The mixture was stirred under a hydrogen atmosphere for 4.5 h
until all starting material disappeared (TLC control). Formation of a
slightly less polar product was observed. The reaction mixture was
filtered through Celite, and the filtrate was concentrated. The residue
was purified by flash chromatography on silica gel. Elution with
chromatography on silica gel. Elution with hexane/Et2O (98:2) gave
1
dienyne 19 (90 mg, 92%): [α]24 −30.7 (c 1.1, CHCl3); H NMR
D
(200 MHz, CDCl3) δ 0.07 and 0.10 (6H and 6H, each s, 4 × SiCH3),
0.56 (6H, q, J = 7.8 Hz, 3 × SiCH2), 0.66 (3H, s, H3-18), 0.889 and
0.894 (9H and 9H, each s, 2 × Si-t-Bu), 0.93 (3H, d, J = 7.4 Hz, H3-
21), 0.94 (9H, t, J = 7.8 Hz, 3 × SiCH2CH3), 1.19 (6H, s, H3-26 and
H3-27), 1.87 and 1.92 (3H and 3H, each br s, CH3-9 and H3-19), 4.08
(1H, br m, H-3α), 4.20 (1H, narr m, H-1β); 13C NMR (50 MHz,
CDCl3) δ −4.6, −4.5, −4.4, −4.1, 7.0, 7.3, 11.3, 18.2, 18.4, 18.9, 19.4,
21.0, 21.3, 25.0, 26.0, 26.1, 28.7, 30.0, 30.2, 31.0, 36.4, 36.5, 36.6, 40.2,
41.6, 42.2, 45.7, 50.8, 55.0, 64.5, 70.3, 73.6, 91.9, 92.5, 116.1, 116.9,
139.7, 140.84; HRMS (ESI) exact mass calcd for C46H86O3Si3Na (M +
Na)+ 793.5783, found 793.5764.
hexane/Et2O (95:5) furnished previtamin 17 (51 mg, 93%): [α]24
+2.6 (c 1.3, CHCl3); H NMR (200 MHz, CDCl3) δ 0.04, 0.05, and
D
1
0.10 (3H, 3H and 6H, each s, 4 × SiCH3), 0.69 (3H, s, H3-18), 0.88
and 0.89 (9H and 9H, each s, 2 × Si-t-Bu), 0.95 (3H, d, J = 6.4 Hz, H3-
21), 1.22 (6H, s, H3-26 and H3-27), 1.44 and 1.65 (3H and 3H, each
br s, CH3-9 and H3-19), 4.05 (1H, br m, H-3α), 4.13 (1H, narr m, H-
1β), 5.75 and 6.00 (1H and 1H, each d, J = 12.2 Hz, H-7 and H-6);
13C NMR (50 MHz, CDCl3) δ −4.8, −4.7, −4.6, −4.2, 11.7, 17.6, 18.0,
18.2, 18.8, 19.7, 20.9, 24.2, 25.9, 26.0, 28.8, 29.2, 29.3, 31.2, 36.2, 36.4,
36.6, 38.7, 41.8, 42.3, 44.4, 51.7, 54.4, 64.9, 71.1, 71.6, 129.1, 129.1,
129.3, 130.6, 131.0, 131.2; UV (EtOH) λmax = 248 nm (ε = 10400);
HRMS (ESI) exact mass calcd for C40H74O3Si2Na (M + Na)+
681.5074, found 681.5093.
1α,25-Dihydroxy-9-methylprevitamin D3 (6). Tetrabutylam-
monium fluoride (1.0 M in THF; 2.34 mL, 2.34 mmol) was added to a
solution of the protected previtamin D analogue 17 (51 mg, 0.078
mmol) in anhydrous THF (5 mL). The reaction mixture was stirred
overnight and quenched by addition of brine, extracted with ethyl
1α-[(tert-Butyldimethylsilyl)oxy]-9-methyl-25-[(triethylsilyl)-
oxy]-previtamin D3 tert-Butyldimethylsilyl Ether (20). Previta-
min 20 was obtained by hydrogenation of the dienyne 19 performed
analogously to the process described above for the preparation of 17.
The product was purified by flash chromatography (hexane) on silica
gel to give previtamin 20 (86 mg, 95%) as a colorless oil: [α]24D + 2.6
1
(c 1.1, CHCl3); H NMR (200 MHz, CDCl3) δ 0.04, 0.05, and 0.10
(3H, 3H and 6H, each s, 4 × SiCH3), 0.56 (6H, q, J = 7.6 Hz, 3 ×
SiCH2), 0.70 (3H, s, H3-18), 0.88 and 0.89 (9H and 9H, each s, 2 × Si-
1448
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450

The Journal of Organic Chemistry
Article
t-Bu), ca. 0.89 (3H, H3-21, overlapped), 0.95 (9H, t, J = 7.6 Hz, 3 ×
SiCH2CH3), 1.19 (6H, s, H3-26 and H3-27), 1.43 and 1.65 (3H and
3H, each br s, CH3-9 and H3-19), 4.05 (1H, br m, H-3α), 4.13 (1H,
narr m, H-1β), 5.75 and 6.00 (1H and 1H, each d, J = 12.2 Hz, H-7
and H-6); 13C NMR (50 MHz, CDCl3) δ −4.6, −4.5, −4.4, −4.0, 7.0,
7.3, 11.8, 17.7, 18.2, 18.4, 18.9, 19.8, 21.0, 24.3, 26.0, 26.2, 28.9, 30.0,
30.2, 31.3, 36.4, 36.6, 36.8, 38.9, 42.0, 42.4, 45.7, 51.9, 54.7, 65.1, 71.8,
73.7, 129.2, 129.2, 129.5, 130.8, 131.2, 131.3; UV (EtOH) λmax = 246
nm (ε = 10400); HRMS (ESI) exact mass calcd for C46H88O3Si3Na
(M + Na)+ 795.5939, found 795.5959.
112.6, 116.5, 125.5, 137.0, 144.0, 146.9; UV (EtOH) λmax = 264 nm (ε
= 16300); HRMS (ESI) exact mass calcd for C28H46O3Na (M + Na)+
453.3345, found 453.3332.
ASSOCIATED CONTENT
■
S
* Supporting Information
General experimental details, spectra (1H and 13C NMR) of all
new compounds, and HPLC chromatograms. This material is
available free of charge via the Internet at http://pubs.acs.org.
1α,25-Dihydroxy-9α-methylvitamin D3 (4), 1α,25-Dihy-
droxy-9-methylene-10(S),19-dihydrovitamin D3 (7), and
1α,25-Dihydroxy-9-methylene-10(R),19-dihydrovitamin D3
(8). Protected from light, a solution of previtamin 6 (28 mg, 0.065
mmol) in isooctane (16 mL) was refluxed for 14 h and then
concentrated. The resulting mixture of isomeric compounds was
separated by HPLC (9.4 mm × 25 cm Eclipse XDB-C18 column, 4
mL/min) using a methanol/water (86:14) solvent system. The 10α-
methyl compound 8 was collected at Rv 32 mL (4.2 mg, 15%), 10β-
methyl compound 7 at Rv 36 mL (7.8 mg, 28%), 9α-methylcalcitriol
(4) at Rv 46 mL (1.7 mg, 6%), and the unreacted previtamin 6 at Rv
48 mL (11.2 mg, 40%).
AUTHOR INFORMATION
Corresponding Author
*E-mail: antonio.mourino@usc.es, rasici@chem.uw.edu.pl.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
U.K. thanks the Foundation for Polish Science; the MPD
Program has been cofinanced by the EU European Regional
Development Fund. R.S. and A.M. thank the Spanish Ministry
of Education and Innovation (MEI) (Project No. SAF2010-
15291) for financial support.
1α,25-Dihydroxy-9α-methylvitamin D3 (4). Final purification of
the vitamin D compound 4 was achieved by HPLC (10 mm × 25 cm
Phenomenex Luna silica column, 4 mL/min) using a hexane/2-
propanol (8:2) solvent system. The vitamin D compound 4 was
1
collected at Rv 50 mL: [α]24 −19.4 (c 0.5, CHCl3); H NMR (500
D
REFERENCES
■
MHz, CDCl3) δ 0.536 (3H, s, H3-18), 0.941 (3H, d, J = 6.4 Hz, H3-
21), 1.076 (3H, d, J = 7.0 Hz, CH3-9α), 1.218 (6H, s, H3-26 and H3-
27), 2.32 (1H, dd, J = 12.9, 6.5 Hz, H-4β), 2.60 (1H, dd, J = 13.0, 3.2
Hz, H-4α), 3.07 (1H, approx quintet, J ≈ 7 Hz, H-9β), 4.23 and 4.44
(1H and 1H, each m, H-3α and H-1β), 5.01 and 5.33 (1H and 1H,
each br s, H2-19), 5.96 and 6.38 (1H and 1H, each dd, J = 11.2, 1.6 Hz,
H-7 and H-6); 13C NMR (125 MHz, CDCl3) δ 11.5, 18.8, 19.9, 20.8,
22.6, 27.6, 29.2, 29.3, 29.4, 30.1, 35.7, 36.1, 36.4, 42.8, 44.4, 45.3, 45.9,
50.8, 56.5, 66.9, 70.8, 71.2, 111.9, 117.1, 124.7, 132.9, 147.4, 147.6; UV
(EtOH) λmax = 265 nm (ε = 17500); HRMS (ESI) exact mass calcd
for C28H46O3Na (M + Na)+ 453.3345, found 453.3346.
(1) (a) Feldman, D., Pike, J. W., Glorieux, F. H., Eds. Vitamin D, 2nd
ed.; Elsevier Academic Press: Burlington, MA, 2005. (b) Feldman, D.,
Pike, J. W., Adams, J. S., Eds. Vitamin D, 3rd ed.; Elsevier Academic
Press: San Diego, CA, 2011.
(2) (a) Pike, J. W.; Meyer, M. B.; Bishop, K. A. Rev. Endocr. Metab.
Dis. 2012, 13, 45−55. (b) Campbell, M. J.; Adorini, L. Expert Opin.
Ther. Targets 2006, 10, 735−748.
(3) Carlberg, C.; Quack, M.; Herdick, M.; Bury, Y.; Polly, P.; Toell,
A. Steroids 2001, 66, 213−221.
(4) (a) Evans, R. M. Science 1988, 240, 889−895. (b) Evans, R. M.
Mol. Endocrinol. 2005, 19, 1429−1438.
1α,25-Dihydroxy-9-methylene-10(S),19-dihydrovitamin D3
(7). Final purification of compound 7 was achieved by HPLC (10
mm × 25 cm Phenomenex Luna silica column, 4 mL/min) using a
(5) Jones, G.; Strugnell, S. A.; DeLuca, H. F. Physiol. Rev. 1998, 78,
1193−1231.
(6) Holick, M. F.; MacLaughlin, J. A.; Doppelt, S. H. Science 1981,
211, 590−593.
hexane/2-propanol (8:2) solvent system. The vitamin D compound 7
1
was collected at Rv 32 mL: [α]24 +72.8 (c 0.9, CHCl3); H NMR
D
(7) Hanewald, K. H.; Rappoldt, M. P.; Roborgh, J. R. Recl. Trav.
Chim. Pays-Bas. 1961, 80, 1003−1014.
(500 MHz, CDCl3) δ 0.637 (3H, s, H3-18), 0.938 (3H, d, J = 6.6 Hz,
H3-21), 1.107 (3H, d, J = 7.6 Hz, H3-19), 1.217 (6H, s, H3-26 and H3-
27), 2.24 (1H, ddd, J = 14.2, 4.7, 2.5 Hz, H-11α), 2.31 (1H, br t, J =
13.2 Hz, H-4β), ca. 2.36 (1H, H-11β, overlapped), 2.38 (1H, dd, J =
13.2, 5.7 Hz, H-4α), 3.02 (1H, br q, J = 7.5 Hz, H-10α), 3.94 (1H, narr
m, H-1β), 3.96 (1H, br m, H-3α), 4.77 and 5.01 [1H and 1H, each t, J
= 2.5 Hz, C(9)CH(Z) and C(9)CH(E)], 5.91 (1H, dd, J = 11.5, 1.8
Hz, H-7), 6.48 (1H, d, J = 11.5 Hz, H-6); 13C NMR (125 MHz,
CDCl3) δ 12.1, 17.2, 19.0, 21.1, 22.6, 28.5, 29.6, 29.6, 33.1, 36.3, 36.6,
37.6, 38.0, 40.8, 42.1, 44.6, 45.8, 56.2, 57.2, 67.6, 71.3, 73.4, 112.5,
116.6, 126.1, 136.6, 144.1, 146.9; UV (EtOH) λmax = 262 nm (ε =
17400); HRMS (ESI) exact mass calcd for C28H46O3Na (M + Na)+
453.3345, found 453.3352.
(8) (a) Woodward, R. B.; Hoffman, R. J. Am. Chem. Soc. 1965, 87,
2511−2513. (b) Woodward, R. B.; Hoffman, R. Angew. Chem. 1969,
81, 797−869.
(9) (a) Havinga, E.; de Kock, R. J.; Rappoldt, M. P. Tetrahedron
1960, 11, 276−284. (b) Havinga, E.; Schlatmann, J. L. M. A.
Tetrahedron 1961, 16, 146−152. (c) Havinga, E. Chimia 1962, 16,
145−151. (d) Schlatmann, J. L. M. A.; Pot, J.; Havinga, E. Recl. Trav.
Chim. Pays-Bas 1964, 83, 1173−1184. (e) Havinga, E. Experientia
1973, 29, 1181−1316.
(10) (a) Akhtar, M.; Gibbons, C. J. Tetrahedron Lett. 1965, 6, 509−
512. (b) Akhtar, M.; Gibbons, C. J. J. Chem. Soc. 1965, 5964−5968.
(11) (a) Sheves, M.; Berman, E.; Mazur, Y.; Zaretskii, Z. J. Am. Chem.
Soc. 1979, 101, 1882−1883. (b) Moriarty, R. M.; Paaren, H. E.
Tetrahedron Lett. 1980, 21, 2389−2392.
1α,25-Dihydroxy-9-methylene-10(R),19-dihydrovitamin D3
(8). Final purification of vitamin 8 was achieved by HPLC (10 mm
× 25 cm Phenomenex Luna silica column, 4 mL/min) using a hexane/
2-propanol (8:2) solvent system. The vitamin D compound 8 was
(12) Spangler, C. W. Chem. Rev. 1976, 76, 187−217.
(13) (a) Hoeger, C. A.; Okamura, W. H. J. Am. Chem. Soc. 1985, 107,
268−270. (b) Hoeger, C. A.; Johnston, A. D.; Okamura, W. H. J. Am.
Chem. Soc. 1987, 109, 4690−4698.
1
collected at Rv 35 mL: [α]24 +118.2 (c 0.7, CHCl3); H NMR (500
D
MHz, CDCl3) δ 0.645 (3H, s, H3-18), 0.941 (3H, d, J = 6.6 Hz, H3-
21), 1.060 (3H, d, J = 7.4 Hz, H3-19), 1.219 (6H, s, H3-26 and H3-27),
2.24 (1H, ddd, J = 14.0, 4.7, 2.5 Hz, H-11α), 2.36 (1H, br m, H-11β),
2.58 (1H, br d, J ≈ 15 Hz, H-4α), 3.27 (1H, m, H-10β), 4.05 (1H, dt, J
= 12.2, 5.2 Hz, H-1β), 4.12 (1H, narr m, H-3α), 4.77 and 5.01 [1H
and 1H, each t, J = 2.4 Hz, C(9)CH(Z) and C(9)CH(E)], 5.95 and
6.34 (1H and 1H, each dd, J = 11.3, 1.6 Hz, H-7 and H-6); 13C NMR
(125 MHz, CDCl3) δ 10.3, 11.9, 19.0, 21.1, 22.6, 28.6, 29.5, 29.6, 33.1,
36.3, 36.4, 36.6, 37.9, 39.1, 40.8, 44.6, 45.8, 56.2, 57.2, 67.5, 68.1, 71.3,
(14) (a) Zhu, G. D.; Okamura, W. H. Endocr. Rev. 1995, 95, 1877−
1957. (b) Nagpal, S.; Na, S. Q.; Rathnachalam, R. Endocr. Rev. 2005,
26, 662−687.
(15) Maestro, M. A.; Sardina, J. F.; Castedo, L.; Mourino, A. J. Org.
̃
Chem. 1991, 56, 3582−3587.
(16) An alternative procedure for such α-methylation described in
the Japanese patent for an analogous MEM-protected hydroxy ketone
1449
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450

The Journal of Organic Chemistry
Article
was significantly less efficient: Shimizu M. 9-Substituted-19-norvitamin
D Derivative. WIPO Patent Application WO/2006/093290.
(17) Sicinski, R. R.; Perlman, K. L.; DeLuca, H. F. J. Med. Chem.
1994, 37, 3730−3738.
(18) (a) Castedo, L.; Mascarenas, J. L.; Mourino, A. Tetrahedron Lett.
̃
̃
1987, 28, 2099−2102. (b) Aurrecoechea, J. M.; Okamura, W. H.
Tetrahedron Lett. 1987, 28, 4947−4950. (c) Okamura, W. H.;
Aurrecoechea, J. M.; Gibbs, R. A.; Norman, A. W. J. Org. Chem.
1989, 54, 4072−4083.
(19) Barton, D. H. R.; Bashiardes, G.; Fourrey, J.-L. Tetrahedron Lett.
1983, 24, 1605−1608.
(20) (a) Castedo, L.; Mourino, A.; Sarandeses, L. Tetrahedron Lett.
̃
1986, 27, 1523−1526. (b) Mascarenas, J. L.; Sarandeses, L. A.;
̃
Castedo, L.; Mourino, A. Tetrahedron 1991, 47, 3485−3489.
̃
(21) It was necessary to use an excess of enyne 10 due to the
concomitant homocoupling process of the A-ring fragment.
(22) Scott, W. J.; Crisp, G. T.; Stille, J. K. J. Am. Chem. Soc. 1984,
106, 4630−4632.
(23) Sheves, M.; Mazur, Y. J. Chem. Soc., Chem. Commun. 1977, 21−
22.
(24) Burgess, E. M.; Penton, H. R., Jr.; Taylor, E. A. J. Org. Chem.
1973, 38, 26−31.
(25) Sicinski, R. R.; Perlman, K. L.; Prahl, J.; Smith, C.; DeLuca, H. F.
J. Med. Chem. 1996, 39, 4497−4506.
(26) (a) Johnson, F.; Malhotra, S. K. J. Am. Chem. Soc. 1965, 87,
5492−5493. (b) Johnson, F. Chem. Rev. 1968, 68, 375−413.
(27) Nasipuri, D. Stereochemistry of Organic Compounds; Wiley
Eastern: New Delhi, 1991; pp 270−271.
(28) Sicinski, R. R.; DeLuca, H. F. Bioorg. Chem. 1994, 22, 150−171.
1450
dx.doi.org/10.1021/jo302513e | J. Org. Chem. 2013, 78, 1444−1450