Synthesis and Biological Activity of 2-Methylene Analogues of Calcitriol and Related Compounds

Article abstract of DOI:10.1021/acs.jmedchem.5b01295

In an attempt to prepare Vitamin D analogues that are superagonists, (20R)- and (20S)-isomers of 1α-hydroxy-2-methyleneVitamin D₃ and 1α,25-dihydroxy-2-methyleneVitamin D₃ have been synthesized. To prepare the desired A-ring dienyne

Full text of DOI:10.1021/acs.jmedchem.5b01295

Article
pubs.acs.org/jmc
Synthesis and Biological Activity of 2‑Methylene Analogues of
Calcitriol and Related Compounds
Izabela K. Sibilska,†,‡ Marcin Szybinski,†,‡ Rafal R. Sicinski,†,‡ Lori A. Plum,† and Hector F. DeLuca*,†
†Department of Biochemistry, University of WisconsinMadison, 433 Babcock Drive, Madison, Wisconsin 53706, United States
‡Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
S
* Supporting Information
ABSTRACT: In an attempt to prepare vitamin D analogues
that are superagonists, (20R)- and (20S)-isomers of 1α-
hydroxy-2-methylenevitamin D3 and 1α,25-dihydroxy-2-
methylenevitamin D3 have been synthesized. To prepare the
desired A-ring dienyne fragment, two different approaches
were used, both starting from the (−)-quinic acid. The
obtained derivative was subsequently coupled with the C,D-
ring enol triflates derived from the corresponding Grundmann
ketones, using the Sonogashira reaction. Moreover, (20R)- and
(20S)-1α,25-dihydroxy-2-methylenevitamin D3 compounds
with an (5E)-configuration were prepared by iodine catalyzed
isomerization. All four 2-methylene analogues of the native hormone were characterized by high in vitro activity. As expected, the
25-desoxy analogues were much less potent. Among the synthesized compounds, two of them, 1α,25-dihydroxy-2-
methylenevitamin D3 and its C-20 epimer, were found to be almost as active as 2-methylene-19-nor-(20S)-1α,25-
dihydroxyvitamin D3 (2MD) on bone but more active in intestine.
However, the lack of the 3β- and especially the 1α-hydroxyl
groups caused a marked decrease in the ability to bind the
vitamin D receptor (VDR). The 1α-hydroxylation of 2,10-
dimethylene compounds 4 and 5 occurs at a rate much slower
than that of 25-hydroxyvitamin D3. Thus, it is suggested that
the next targets in our structure−activity studies should include
compounds that possess both the A-ring hydroxyls (1α and 3β)
and both exomethylene substituents at C-2 and C-10. This
paper reports the preparation and testing of these compounds,
8−13.13
INTRODUCTION
■
The most active metabolite of vitamin D3, 1α,25-dihydrox-
yvitamin D3 [calcitriol, 1α,25-(OH)2D3, 1, Figure 1) plays a
crucial role in calcium and phosphate homeostasis.1 In addition,
the physiological role of calcitriol in living organisms is much
broader than previously thought and includes the regulation of
cellular growth, cell differentiation, and immunomodulation.2
This has resulted in a search for analogues that target these
roles with reduced calcemic activity.3
By moving the exomethylene substituent of calcitriol from C-
10 to C-2 (compound 2) there is a marked increase in bone
calcium mobilization activity.4 This effect was even more
pronounced when the configuration was changed from 20R to
20S. Further, this analogue also greatly increased bone
formation in vitro5 as well as in the ovariectomized (OVX)
rat model.6 Moreover in a clinical trial, compound 3 increased
bone turnover in postmenopausal women.7 These findings
stimulated synthesis of several other 2-methylene-19-norvita-
min D analogues acting as possible agents for the treatment of
osteoporosis.8 To explore the surprising high biological activity
of 2MD, 1-desoxy9 and 3-desoxy analogues of 2MD were
synthesized.10 We also prepared the (20R)- and (20S)-25-
hydroxy-2-methylenevitamin D3 compounds 4 and 511 and the
(20R) and (20S) isomers of 3-desoxy-1α,25-dihydroxy-2-
methylenevitamin D3 (6 and 7).12 Biological activities of
these analogues were compared with previously obtained 2-
methylene-substituted vitamin D compounds. Compounds 4−
7, possessing both exomethylene moieties at C-2 and C-10,
were characterized by pronounced in vivo calcemic activity.
RESULTS AND DISCUSSION
■
Chemistry. Since the bicyclic vinyl triflates 15−17 were
known compounds and 18 could be easily prepared from the
respective Grundmann ketone by the same method, we
concentrated our efforts on the synthesis of the required A-
ring dienyne 14. Two alternative synthetic routes have been
elaborated, both starting from the commercially available
(−)-(1S,3R,4S,5R)-quinic acid (19).
First synthetic approach began with the silylation of the
secondary hydroxyl in the ester 20 (Scheme 1) easily accessible
from 19.14 Treatment of the obtained hydroxy ester 21 with
sulfuryl chloride in pyridine provided a single dehydration
product 22. The structure of the 22 assignment was based on
previous experience in similar systems.15 Introduction of the
methyl group into β-position of unsaturated ester 22 was
Received: August 20, 2015
© XXXX American Chemical Society
A
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Figure 1. Chemical structure of 1α,25-dihydroxyvitamin D3 (calcitriol, 1), the previously synthesized 2-methylene compounds 2−7, 2-methylene
analogues described in this work (8−13), and the building blocks for their synthesis.
a
Scheme 1
a
(a) TMS-Cl, TEA, CH2Cl2 (21: 97%, 24: 88%, 28: 88%); (b) SO2Cl2, pyridine; then MeOH, CH2Cl2, 68%; (c) CH2N2, Et2O, 95%; (d) (i) DMF,
125 °C; (ii) DIBALH, CH2Cl2/toluene, chrom. separation (25, 32%; 26, 32%; two steps); (e) citric acid, MeOH, 89%; (f) Dess−Martin
periodinane, CH2Cl2, 82%; (g) (i) Ph3P+CH3Br−, n-BuLi, THF; (ii) 5% HCl, 76% (two steps); (h) PDC, CH2Cl2, 79%; (i) n-BuLi, TMSCHN2,
THF, 82%.
achieved by the method described by Desmaele and Tanier.16
Treatment of 22 with diazomethane resulted in formation of
bicyclic adduct 23 as a product of 1,3-dipolar cycloaddition.
The observed regioselectivity of the reaction was expected;17
however, high stereoselectivity of this process was somewhat
surprising. Inspection of 1H NMR spectrum of the product and
comparison of its vicinal coupling constants with those
calculated by molecular modeling (Figure 2a) allowed us to
propose adduct structure resulting from an attack of the
diazomethane from the side of the allylic OTBS group. The
hydroxyl group in 23 was then protected as a TMS ether and
the silylated adduct 24 subjected to thermolysis in DMF.
Unfortunately, the outcome of this process was rather
disappointing because two products, resulting from nitrogen
extrusion, were formed in comparable quantities, and their
separation was possible only after the following reduction step.
In addition to the desired allylic alcohol 26, the bicyclic product
1
25 was also isolated and its structure was established by H
NMR (Figure 2b). After manipulation of protective groups, 26
was converted to the isomeric alcohol 28, which was oxidized
with Dess−Martin periodinane. The obtained ketone 29 was
then subjected to Wittig methylenation and hydrolysis.
Oxidation of the formed allylic alcohol 30 with PDC afforded
the aldehyde 31 that reacted with the anion of trimethylsi-
lyldiazomethane leading to the target dienyne 14.
An alternative route to the A-ring fragment 14 started with
the keto lactone 32 (Scheme 2) prepared from the quinic acid
19 by the described method.18 Wittig reaction introduced the
B
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Figure 2. Preferred, energy-minimized (HyperChem) conformations of bicyclic diazomethane adducts 23 (a) and 37 (c) and bicyclic products 25
(b) and 38 (d) resulting from the pyrolysis of the adducts and the following reduction. The most informative 1H−1H constants are listed; values of
calculated couplings (PC MODEL) are enclosed in parentheses.
exomethylene substituent at the early stage of synthesis, and
methanolysis of 33 gave the dihydroxy ester 34. As a result of
the symmetrical substitution of its cyclohexane ring, the
subsequent dehydration process with Martin sulfurane
furnished a single elimination product 36 from 35. 1,3-Dipolar
cycloaddition of diazomethane to the unsaturated ester 36
proved to be highly regio- and stereoselective, occurring solely
from the side of an allylic OTBS substituent. Inspection of the
1H NMR spectra of the formed adduct 37, supported by
molecular mechanics calculations, allowed us to establish its
structure and preferred conformation (Figure 2c). The
subsequent thermolysis process of 37 followed by DIBALH
reduction provided two isomeric compounds. However, the
yield of the desired allylic alcohol 30, a direct precursor of the
A-ring fragment 14, was significantly higher (60%) than that of
the minor bicyclic product 38 (34%; Figure 3d). A comparison
of the two synthetic paths described above (Schemes 1 and 2)
indicates that the overall yield of 30 (from quinic acid) is
almost twice as high in the latter method (5.5% versus 10%).
The A-ring building block 14 was than coupled with the C,D-
ring vinyl triflates 15−18, obtained from the corresponding
Grundmann ketones.4,19 Three former hydrindane compounds
were described in literature,12,20,21 and 18 was prepared in an
analogous fashion (Supporting Information). Thus, Sonoga-
shira reaction of dienyne 14 with the vinyl triflate 15 carried out
using reactions described by Mourino21 furnished the expected
trienyne 39 (Scheme 3) in which the triple bond was selectively
hydrogenated by a Lindlar catalyst poisoned with quinoline.
Thermal rearrangement of the obtained previtamin D analogue
43 afforded the protected vitamin D compound 47 in
quantitative yield. Removal of the three silyl protecting groups
was less efficient but provided the target 2-methylene-1α,25-
(OH)2D3 (8). The Sonogashira protocol was also applied to
other coupling reactions between dienyne 14 and the vinyl
triflates 16−18. The obtained trienynes 40−42 were converted
to the respective final vitamin D analogues 9−11 as described
above. For the preparation of the 5E-compounds 12 and 13,
the well-known iodine-catalyzed isomerization22 was used
(Scheme 4).
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a
Scheme 2
a
(a) Ph3P+CH3Br−, t-BuOK, THF, 73%; (b) CH3OMe, MeOH, 79%; (c) TBSOTf, 2,6-lutidine, CH2Cl2, 95%; (d) Martin sulfurane, CCl4, 90%; (e)
CH2N2, Et2O, 99%; (f) (i) DMF, 125 °C; (ii) DIBALH, CH2Cl2/toluene, chrom separation (30, 60%; 38, 34%; two steps).
The results of in vivo testing of these analogues clearly
indicate that 1α,25-dihydroxy-2-methylenevitamin D3 (8) and
its isomer 9 with an “unnatural” (20S)-configuration were the
most active in raising of serum calcium at the expense of bone
(Figures 3 and 4). Their 25-desoxy counterparts 10 and 11
were less active but nevertheless more active than calcitriol.
Interestingly, the (5E)-configuration found in analogues 12 and
13 resulted in decreased activity on bone calcium mobilization
(serum calcium). The same pattern of activity was observed for
intestinal calcium transport (Figures 5 and 6).
CONCLUSIONS
■
Figure 3. Bone calcium mobilization of 1α,25-(OH)2D3 (1), its
synthesized 2-methylene analogues 8 and 12, and 2-methylene
Two alternative synthetic paths to prepare 2-methylene-
substituted vitamin D compounds were used, both providing
analogue of 1α-OH-D3 (10).
the desired target compounds which were then tested
biologically (Table 1, Figures 3−6). Introduction of an
additional A-ring exomethylene group at C-2 in 1α,25-
(OH)2D3 (1) increased both bone calcium mobilization and
intestinal calcium transport in vivo without any change in in
vitro tests. The (20S)-analogues with both a 2- and 10-
methylene group (compound 9) or pseudo-4-methylene group
(compound 13) have VDR binding and HL-60 activity similar
to 2MD, whereas their transcriptional activity was decreased 3
and 6 times, respectively, as compared to 3. The bone calcium
mobilization activity of (20S)-1α,25-dihydroxy-2-methylene-
vitamin D3 (9) was found to be slightly lower than its 19-nor
analogue 3, similar to its 20R-epimer 8 and approximately an
order of magnitude higher than calcitriol. However, in the
intestine, both compounds 8 and 9 were either equal or slightly
more potent than 1 and 3. Strong calcemic activity of analogues
10 and 11 lacking the 25-hydroxyl suggests their efficient
enzymatic hydroxylation in vivo. Because of their considerable
in vivo activity, these compounds might be considered potential
prodrugs.
Biological Evaluation. The affinity of the analogues for the
full-length recombinant rat receptor was compared to that of
1α,25-(OH)2D3 (1) and 2MD (3). It was established (Table 1)
that 1α,25-dihydroxy-2-methylenevitamin D3 (8) as well as its
(20S)- and (5E,20S)-isomers bound the VDR slightly more
effectively as the natural hormone 1, whereas (5E)-compound
12 had 2.5-fold higher affinity for the receptor. Not
unexpectedly, the lack of 25-hydroxyl in the analogues 10
and 11 resulted in much lower binding ability, decreased by 2
orders of magnitude from 1.
With the exception of 10, the obtained compounds were
strongly antiproliferative. Analogue 9 and its (5E)-counterpart
13 were either as effective or more effective than 2MD in
inducing HL-60 cells to differentiate into monocytes.
The activity of the synthesized compounds in inducing
transcription of a vitamin D target gene was examined using the
1,25(OH)2D3 hydroxylase (CYP24A1) promoter. Analogues 8
and 12, with the natural configuration at C-20, had transcrip-
tional activity equal to that of 1α,25-(OH)2D3 (1). 25-Desoxy
compounds 10 and 11 also induced a dose-dependent
activation of the CYP24A1 gene but were less active than 1
by an order of magnitude. As in the previous assay of all tested
compounds, the most pronounced activity was exhibited by
(20S)-analogues 9 and 13.
EXPERIMENTAL SECTION
■
Chemistry. Optical rotations were measured in chloroform using
PerkinElmer models 241 and 343 polarimeters at 22 °C. Ultraviolet
(UV) absorption spectra were obtained on a Shimadzu UV-1800 UV
spectrophotometer in 100% EtOH. All nuclear magnetic resonance
D
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a
Scheme 3
a
(a) (PPh3)2Pd(OAc)2, CuI, Et2NH, DMF (39, 92%; 40, 54%; 41, 84%; 42, 98%); (b) Lindlar cat., H2, hexane (43, 70%; 44, 84%; 45, 83%; 46,
85%); (c) 65 °C, hexane (47, 100%; 48, 70%; 49, 70%; 50, 91%); (d) TBAF, THF (8, 40%; 9, 16%; 10, 40%; 11, 46%).
starting hydroxy ester 2014 and lactone 3218 were synthesized from
(−)-quinic acid (19).
a
Scheme 4
(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-
[(trimethylsilyl)oxy]cyclohexanecarboxylic Acid Methyl Ester
(21). To a stirred solution of the dihydroxy ester 20 (1.03 g, 2.4
mmol) in anhydrous methylene chloride (5 mL) and triethylamine
(600 μL) was added chlorotrimethylsilane (490 μL, 3.9 mmol) at 0
°C. After 2 h the reaction was quenched with saturated NaHCO3 and
extracted with methylene chloride. The organic phase was washed with
brine, dried (MgSO4), and concentrated. The residue was purified by
column chromatography on silica using hexane/ethyl acetate (9:1) to
give protected compound 21 (1.17 g, 97%) as a colorless oil.
(3R,4S,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-hydroxycy-
clohex-1-enecarboxylic Acid Methyl Ester (22). To a stirred
a
(a) I2, hν, Et2O (12, 65%; 13, 64%).
solution of hydroxy ester 21 (11.23 g, 22.2 mmol) in anhydrous
methylene chloride (100 mL) and pyridine (23 mL) was dropwise
added sulfuryl chloride (6.8 mL, 84 mmol) at −78 °C under argon.
spectra were recorded in deuteriochloroform using Varian Unity Plus
(200 MHz), Bruker DMX-400 (400 MHz), Bruker DMX-500 (500
MHz). COSY spectra, spin decoupling as well as NOE, DEPT 90, and
DEPT 135 experiments were used to assign particular signals in the 1H
and 13C NMR spectra. Chemical shifts (δ) are reported in parts per
million relative to (CH3)4Si (δ 0.00) as an internal standard.
Abbreviations used are singlet (s), doublet (d), triplet (t), quartet
(q), multiplet (m). High resolution mass spectra were registered on
LCT (TOF) or Mass Quattro LC spectrometer. High-performance
liquid chromatography (HPLC) was performed on a Waters
Associates liquid chromatograph equipped with a model 6000A
solvent delivery system, model U6K Universal injector, and model 486
tunable absorbance detectors. Solvents were dried and distilled
following standard procedures.
The white suspension was stirred for 5 h at this temperature, and then
methanol (12 mL) was dropwise added. The mixture was allowed to
warm up to room temperature, and stirring was continued for 17 h.
The reaction was quenched with brine and extracted with diethyl
ether. The organic phase was dried (MgSO4) and evaporated. The
residue was purified by column chromatography on silica using
hexane/ethyl acetate (95:5) to afford an oily unsaturated ester 22
(1.17 g, 97%).
(3aR,4R,5S,6R,7aR)-4,6-Bis[(tert-butyldimethylsilyl)oxy]-5-
hydroxy-3,3a,4,5,6,7-hexahydroindazole-7a-carboxylic Acid
Methyl Ester (23). To a solution of unsaturated ester 22 (1.94 g,
4.66 mmol) in diethyl ether (10 mL) was added an ether solution of
diazomethane [20 mL (prepared according to the procedure of
Arndt)].24 The mixture was stirred at room temperature for 24 h in
the darkness. The solvent was evaporated and the crude product was
purified by column chromatography on silica using hexane/ethyl
acetate (92:8) to give a pale yellow, oily adduct 23 (2.03 g, 95%).
(3aR,4R,5S,6R,7aR)-4,6-Bis[(tert-butyldimethylsilyl)oxy]-5-
[(trimethylsilyl)oxy]-3,3a,4,5,6,7-hexahydroindazole-7a-car-
boxylic Acid Methyl Ester (24). Silylation of bicyclic compound 23
was performed according to procedure described for compound 20;
the reaction was carried out overnight at room temperature.
Purification of the crude product by column chromatography on silica
The purity of final compounds was determined by HPLC, indicating
at least 99% purity. Two HPLC columns (9.4 mm × 25 cm Zorbax-Sil
and 9.4 mm × 25 cm Zorbax Eclipse XDB-C18) were used (Table 2 in
Supporting Information). The purity and identity of the synthesized
1
vitamins were confirmed by inspection of their H NMR and high-
resolution mass spectra.
The known vinyl triflates 15,20 16,12 and 1721 were obtained
according to the procedure of De Clercq et al.;23 an analogous method
was used for the preparation of the (20S)-trilate 18 (Supporting
Information) from the corresponding Grundmann ketone. The
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a
b
c
Table 1. Relative VDR Binding Activities, HL-60 Differentiating Activities, and Transcriptional Activities of the Vitamin D
Hormone (1), 2MD (3), and the Vitamin D Analogues 8−13
a
Competitive binding of 1α,25-(OH)2D3 (1) and the synthesized vitamin D analogues to the full-length recombinant rat vitamin D receptor. The
experiments were carried out in duplicate on two different occasions. The Ki values are derived from the dose−response curves and represent the
inhibition constant when radiolabeled 1α,25-(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used. The numbers shown in the table are expressed
b
as the average ratio of the 1α,25-(OH)2D3 Ki to the Ki for the analogue. Induction of differentiation of HL-60 promyelocytes to monocytes by
1α,25-(OH)2D3 (1) and the synthesized vitamin D analogues. Differentiation state was determined by measuring the percentage of cells reducing
nitro blue tetrazolium (NBT). The experiment was repeated in duplicate two times. The ED50 values are derived from the dose−response curves and
represent the analogue concentration capable of inducing 50% maturation. The numbers shown in the table are expressed as the average ratio of the
c
1α,25-(OH)2D3 ED50 to the ED50 for the analogue. Transcriptional assay in rat osteosarcoma cells stably transfected with a 24-hydroxylase gene
reporter plasmid. The ED50 values are derived from dose−response curves and represent the analogue concentration capable of increasing the
luciferase activity by 50%. The numbers shown in the table are expressed as the average ratio of the 1α,25-(OH)2D3 ED50 to the ED50 for the
analogue.
Figure 5. Intestinal calcium transport of 1α,25-(OH)2D3 (1), its
synthesized 2-methylene analogues 8 and 12, and 2-methylene
analogue of 1α-OH-D3 (10).
Figure 4. Bone calcium mobilization of 1α,25-(OH)2D3 (1), its
synthesized 2-methylene analogues 9 and 13, and 2-methylene
analogue of (20S)-1α-OH-D3 (11).
using hexane/ethyl acetate (95:5) gave silylated product 24 (102 mg,
88%) as a pale yellow oil.
[(1′S,3′R,4′S,′5R,6′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-
4′-[(trimethylsilyl)oxy]bicyclo[4.1.0]hept-1-yl]methanol (25)
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room temperature overnight. The mixture was poured into 5% HCl
and extracted with ethyl acetate. The organic layer was washed with
saturated NaHCO3 and brine, dried (MgSO4), and evaporated to give
an orange oily residue which was applied on a silica Sep-Pak cartridge.
Elution with hexane/ethyl acetate (95:5) gave pure alcohol 30 (81 mg,
76%) as a colorless oil.
(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-2-methyl-4-
methylenecyclohex-1-enecarbaldehyde (31). The mixture of
alcohol 30 (16 mg, 40.2 μmol) and pyridinium dichromate (48.5 mg,
225.1 μmol) in anhydrous methylene chloride (0.7 mL) was stirred
vigorously at room temperature for 4 h. The reaction mixture was then
filtered through a pad of Celite (washed with methylene chloride), and
the solvents were removed under reduced pressure. The crude product
was applied on a silica Sep-Pak cartridge and eluted with hexane/
diethyl ether (98:2) to yield the aldehyde 31 (12.6 mg, 79%) as a
colorless oil.
Figure 6. Intestinal calcium transport of 1α,25-(OH)2D3 (1), its
synthesized 2-methylene analogues 9 and 13, and 2-methylene
analogue of (20S)-1α-OH-D3 (11).
(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-ethynyl-2-
methyl-4-methylenecyclohexene (14). n-Butyllithium (1.6 M in
hexanes; 25.5 μL, 40.8 μmol) was added to a solution of
(trimethylsilyl)diazomethane (2.0 M in hexane, 19.5 μL, 39 μmol)
in anhydrous THF (50 μL) at −78 °C under argon, and a solution of
aldehyde 31 (12.6 mg, 31.8 μmol) in dry THF (100 μL + 50 μL) was
added via cannula. After 1 h the cooling bath was removed and stirring
was continued at room temperature overnight. Water was added, and
the mixture was extracted with hexane, dried (Na2SO4), and
concentrated. The crude product was applied on a silica Sep-Pak
cartridge and eluted with hexane to afford dienyne 14 (10 mg, 82%).
(1R,3R,5R)-1-Acetoxy-3-[(tert-butyldimethylsilyl)oxy]-4-
methylene-6-oxabicyclo[3.2.1]octan-7-one (33). A solution of
potassium tert-butoxide in THF (1.0 M; 746 μL, 746 μmol) was added
dropwise to a stirred suspension of methyltriphenylphosphonium
bromide (280 mg, 784.6 μmol) in anhydrous THF (5.5 mL) at 0 °C.
The mixture was warmed up to room temperature and stirred for
additional 10 min. A solution of ketone 32 (126 mg, 382.7 μmol) in
THF (1.6 mL) was added via cannula, and stirring was continued at
room temperature for 1 h. Water was added, and the mixture was
extracted with ethyl acetate, dried (MgSO4), and concentrated. The
residue was applied on a silica Sep-Pak cartridge (5 g) and eluted with
hexane/ethyl acetate (95:5) to afford compound 33 (91 mg, 73%).
(3R,5R)-5-[(tert-Butyldimethylsilyl)oxy]-1,3-dihydroxy-4-
methylenecyclohexanecarboxylic Acid Methyl Ester (34). A
solution of lactone 33 (330 mg, 1.01 mmol) was vigorously stirred in
methanolic sodium methoxide solution (0.04 M; 10 mL, 0.4 mmol) at
room temperature for 17 h under argon. Water was added, and the
mixture was extracted with ethyl acetate, dried (Na2SO4), and
concentrated. The residue was applied on a silica Sep-Pak cartridge
(5 g) and eluted with hexane/ethyl acetate (7:3) to give the diol 34
(253 mg, 79%) as a colorless oil.
(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-1-hydroxy-4-
methylenecyclohexanecarboxylic Acid Methyl Ester (35). 2,6-
Lutidine (191 μL, 1.65 mmol) was dropwise added to a stirred
solution of the diol 34 (274 mg, 865.8 μmol) in anhydrous methylene
chloride (4.5 mL) at −40 °C followed by tert-butyldimethylsilyl
trifluoromethanesulfonate (300 μL, 1.3 mmol). The stirring was
continued at −40 °C for 1 h, and saturated NaHCO3 was added.
Cooling bath was removed, and the reaction mixture was allowed to
warm up slowly to room temperature. The mixture was extracted with
methylene chloride, and combined organic layers were washed with
5% HCl and water, dried (Na2SO4), and concentrated. The residue
was applied on a silica Sep-Pak cartridge (10 g) and eluted with
hexane/ethyl acetate (93:7) to give compound 35 (353.5 mg, 95%) as
a colorless oil.
and [(1′S,3′R,4′S,′5R,6′R)-3′,5′-Bis[(tert-butyldimethylsilyl)-
oxy]-4′-[(trimethylsilyl)oxy]bicyclo[4.1.0]hept-1-yl]methanol
(25) and [(3′R,4′S,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-2′-
methyl-4′-[(trimethylsilyl)oxy]cyclohex-1′-enyl]methanol (26).
A solution of compound 24 (118 mg, 0.22 mmol) in anhydrous DMF
(3 mL) was heated at 125 °C under argon. After 16 h solvent was
removed. The crude product was dissolved in methylene chloride (2.5
mL) and cooled to −78 °C. Diisobutylaluminum hydride (1 M in
methylene chloride; 1 mL, 1 mmol) was added to the mixture, and
stirring was continued for 1 h. The reaction was quenched with
saturated NH4Cl and 1 M potassium−sodium tartrate and extracted
with methylene chloride. The solvent was evaporated and the crude
product was purified by column chromatography on silica using
hexane/diethyl ether/ethyl acetate (95:4:1) to give semicrystalline
alcohols 25 (34 mg, 32%) and 26 (34 mg, 32%).
(1S,2R,6R)-2,6-Bis[(tert-butyldimethylsilyl)oxy]-4-hydroxy-
methyl-3-methylcyclohex-3-enol (27). To a stirred solution of
alcohol 26 (131 mg, 0.28 mmol) in methanol (12 mL) was added
citric acid hydrate (240 mg, 1.11 mmol). The reaction was continued
for 3 h, solvent was evaporated, saturated NaHCO3 was added to the
residue, and the mixture was extracted with ethyl acetate. The organic
phase was dried (MgSO4) and evaporated. The crude product was
purified by column chromatography on silica using hexane/ethyl
acetate (9:1) to afford pure semicrystalline diol 27 (100 mg, 89%).
(1S,2R,6R)-2,6-Bis[(tert-butyldimethylsilyl)oxy]-3-methyl-4-
[(trimethylsilyl)oxy]methylcyclohex-3-enol (28). Silylation of
compound 27 (80 mg, 0.20 mmol) was performed according to
procedure described for compound 20; the reaction was carried out at
−78 °C for 30 min. Purification of the crude product by column
chromatography on silica using hexane/ethyl acetate (95:5) gave
silylated product 28 (83 mg, 88%) as a pale yellow oil.
(1S,2R,6R)-2,6-Bis[(tert-butyldimethylsilyl)oxy]-3-methyl-4-
[(trimethylsilyl)oxy]methylcyclohex-3-enone (29). Dess−Martin
periodinane (1.58 g, 3.73 mmol) was added to a solution of alcohol 28
(1.178 g, 2.49 mmol) in anhydrous methylene chloride (60 mL). The
mixture was stirred for 1 h at room temperature under argon
atmosphere. Then reaction was quenched with saturated Na2S2O3 and
saturated NaHCO3. The mixture was extracted with methylene
chloride, and the organic phase was dried (MgSO4) and evaporated.
The residue was purified by column chromatography on silica using
hexane/ethyl acetate (95:5) to afford ketone 29 (1.05 g, 89%) as a
colorless oil.
[(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-2-methyl-4-
methylene-cyclohex-1-enyl]methanol (30). n-Butyllithium (1.6
M in hexane; 0.6 mL, 0.96 mmol) was added dropwise to
methyltriphenylphosphonium bromide (190 mg, 0.53 mmol) in
anhydrous THF (3 mL) at −78 °C. After 15 min another portion
of phosphonium salt (190 mg, 0.53 mmol) was added, and the
solution was stirred at 0 °C for 40 min. The orange-red mixture was
then cooled to −78 °C and siphoned to the precooled (−78 °C)
solution of the ketone 29 (127 mg, 0.27 mmol) in anhydrous THF (1
mL). The reaction mixture was stirred at −78 °C for 2 h and then at
(3R,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-methylene-
cyclohex-1-enecarboxylic Acid Methyl Ester (36). To a stirred
solution of alcohol 35 (326 mg, 756.8 μmol) in anhydrous carbon
tetrachloride (8.2 mL) was added a solution of bis[α,α-bis-
(trifluoromethyl)benzyloxy]diphenylsulfur (752 mg, 1.12 mmol) in
anhydrous carbon tetrachloride (6 mL) at room temperature under
argon. Reaction was stirred for 30 min, water was added, and the
mixture was extracted with methylene chloride. The organic phase was
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Article
dried (Na2SO4) and concentrated. The resulting residue was applied
on a silica Sep-Pak cartridge (5 g) and eluted with hexane/diethyl
ether (98:2) to give the desired product contaminated by dehydrating
reagent. Further purification on preparative TLC plates (silica gel
60F254, 20 cm × 20 cm, layer thickness 250 nm) using hexane/diethyl
ether (92:8) afforded unsaturated ester 36 (276 mg, 90%) as a
colorless oil.
(3aR,4R,6R,7aR)-4,6-Bis[(tert-butyldimethylsilyl)oxy]-5-
methylene-3,3a,4,5,6,7-hexahydroindazole-7a-carboxylic Acid
Methyl Ester (37). Solution of diazomethane in diethyl ether [2.7 mL
(prepared according to the procedure of Arndt)]24 was added to a
solution of the ester 36 (264 mg, 639.7 μmol) in anhydrous ethyl
ether (1 mL) at room temperature. Reaction mixture was protected
from light and stirred for 2 h. Solvent was evaporated, a residue
dissolved in hexane, applied on a silica Sep-Pak cartridge (5 g), and
eluted with hexane/ethyl acetate (97:3) to give bicyclic adduct 37 (288
mg, 99%) as colorless oil.
[(3′R,5′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-2′-methyl-
4′-methylenecyclohex-1′-enyl]methanol (30) and
[(1′S,3′R,4′S,′5R,6′R)-3′,5′-Bis[(tert-butyldimethylsilyl)oxy]-4′-
[(trimethylsilyl)oxy]bicyclo[4.1.0]hept-1-yl]methanol (38). A
solution of compound 37 (39 mg, 162.8 μmol) in freshly distilled
anhydrous DMF (1.7 mL) was stirred at 125 °C for 6 h under argon.
Heating bath was removed, water was added, and the mixture was
extracted with hexane, dried (Na2SO4), and concentrated. The crude
product was applied on a silica Sep-Pak cartridge (2 g) and eluted with
hexane/diethyl ether (97:3). Removal of the solvents gave an oily
residue (25 mg) that was dissolved in toluene/methylene chloride
(2:1, 3 mL). To this solution diisobutylaluminum hydride (1.0 M in
toluene; 260 μL, 260 μmmol) was slowly added at −78 °C under
argon and stirred for 2 h. The mixture was quenched by a slow
addition of potassium−sodium tartrate (2 N, 4 mL), aqueous HCl (2
N, 4 mL), and H2O (16 mL) and extracted with ethyl acetate. The
combined organic layers were washed with brine, dried (MgSO4), and
concentrated. The residue was applied on a silica Sep-Pak cartridge (2
g) and eluted with hexane/ethyl acetate (98:2) to give the allylic
alcohol 30 (14 mg, 60%) and bicyclic product 38 (8 mg, 34%).
1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien-6-yne (39).
To a solution of dienyne 14 (8 mg, 20.4 μmol) and triflate 15 (8.4
mg, 15.9 μmol) in anhydrous DMF (200 μL) were added CuI (0.45
mg, 2.37 μmol), (PPh3)2Pd(OAc)2 (0.34 mg, 0.45 μmol), and Et2NH
(159 μL) at room temperature under argon. After 45 min the mixture
turned deep reddish-brown. Water was added, and the mixture was
extracted with hexane, dried (MgSO4), and concentrated. The residue
was applied on a silica Sep-Pak cartridge (2 g) and eluted with hexane
to afford trienyne 39 (8.3 mg, 92%) and recovered dienyne 14 (2.2
mg).
(20S)-1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-2-methylene-
25-[(triethylsilyl)oxy]-9,10-secocholesta-5(10),8-dien-6-yne
(40). Sonogashira reaction of dienyne 14 and triflate 16, performed
according to the procedure described above for the coupling of 14 and
15, gave the trienyne 40 (54%).
1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-2-methylene-9,10-se-
cocholesta-5(10),8-dien-6-yne (41). Sonogashira reaction of triflate
17 and the dienyne 14, performed analogously as described above for
the coupling of 14 and 15, gave trienyne 41 (84%).
(20S)-1α,3β-Bis[(tert-butyldimethylsilyl)oxy]-2-methylene-
9,10-secocholesta-5(10),8-dien-6-yne (42). Sonogashira reaction
of the triflate 18 and the dienyne 14 was performed analogously as
described above for the coupling of 14 and 15 to afford trienyne 42
(98%).
1α-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]vitamin D3 tert-Butyldimethylsilyl Ether (47).
To a solution of the trienyne 39 (8.3 mg, 10.8 μmol) in hexane (3
mL) and quinoline (2 μL) was added Lindlar catalyst (25 mg), and the
mixture was stirred at room temperature under a positive pressure of
hydrogen. Lindlar catalyst was added twice during 2.5 h (in 20 mg
portions) and then the mixture was applied on a silica Sep-Pak
cartridge (2 g) and eluted with hexane/ether (98:2) to give the
silylated previtamin 43 (5.8 mg, 70%). The previtamin was then
dissolved in anhydrous hexane (3 mL) and stirred at 60 °C for 14 h
under argon. Solvent was evaporated and residue was applied on a
silica Sep-Pak cartridge (2 g) and eluted with hexane/diethyl ether
(99.6:0.4) to give protected vitamin D compound 47 (5.8 mg, 100%).
(20S)-1α-[(tert-Butyldimethylsilyl)oxy]-2-methylene-25-
[(triethylsilyl)oxy]vitamin D3 tert-Butyldimethylsilyl Ether (48).
Hydrogenation of trienyne 40, performed according to the procedure
described above for 39, gave silylated previtamin 44 (84%).
Compound 44 was then subjected to the analogously performed
thermal isomerization to give protected vitamin 48 (70%).
1α-[(tert-Butyldimethylsilyl)oxy]-2-methylenevitamin D3
tert-Butyldimethylsilyl Ether (49). Hydrogenation of trienyne 41
was performed analogously as described above for 39. The obtained
silylated previtamin 45 (83%) was then subjected to the thermal
isomerization to give protected vitamin 49 (70%).
(20S)-1α-[(tert-Butyldimethylsilyl)oxy]-2-methylenevitamin
D3 tert-Butyldimethylsilyl Ether (50). Hydrogenation of trienyne
42 was performed analogously as described above for 39. The obtained
silylated previtamin 46 (85%) was then subjected to the thermal
isomerization to afford protected vitamin 50 (91%).
1α,25-Dihydroxy-2-methylenevitamin D3 (8). To a solution of
protected vitamin 47 (5.8 mg, 7.5 μmol) in THF (1 mL) was added
tetrabutylammonium fluoride (1.0 M in THF; 450 μL, 450 μmol) at
room temperature under argon. The stirring was continued for 20 h,
brine was added, and the mixture was extracted with ethyl acetate. The
organic extracts were dried (MgSO4) and evaporated. The residue was
purified by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min)
using hexane/2-propanol (9:1) solvent system; compound 8 (1.28 mg,
40%) was collected at RV = 36 mL. Analytical sample of the vitamin
was obtained after reversed-phase HPLC (9.4 mm × 25 cm Zorbax
Eclipse XDB-C18 column, 4 mL/min) using methanol/water (88:12)
solvent system (RV = 33 mL).
(20S)-1α,25-Dihydroxy-2-methylenevitamin D3 (9). Treat-
ment of protected vitamin 48 with TBAF, performed according to
the procedure described above for 47, gave a product that was purified
by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min) using
hexane/2-propanol (92:8) solvent system; vitamin 9 (16%) was
collected at RV = 36 mL. Analytical sample of the vitamin was obtained
after reversed-phase HPLC (9.4 mm × 25 cm Zorbax Eclipse XDB-
C18 column, 4 mL/min) using methanol/water (88:12) solvent
system (RV = 30 mL).
1α-Hydroxy-2-methylenevitamin D3 (10). Hydroxyl deprotec-
tion of silylated vitamin 49 was performed analogously as described
above for 47. The obtained product was purified by HPLC (9.4 mm ×
25 cm Zorbax-Sil column, 4 mL/min) using hexane/2-propanol (95:5)
solvent system; vitamin 10 (1.8 mg, 40%) was collected at RV = 34 mL.
Analytical sample of the vitamin was obtained after reversed-phase
HPLC (9.4 mm × 25 cm Zorbax Eclipse XDB-C18 column, 4 mL/
min) using methanol/water (97:3) solvent system (RV = 40 mL).
(20S)-1α-Hydroxy-2-methylenevitamin D3 (11). Hydroxyl
deprotection of protected vitamin 50 (11 mg, 17.2 μmol) was
performed analogously as described above for 47. The product was
purified by HPLC (9.4 mm × 25 cm Zorbax-Sil column, 4 mL/min)
using hexane/2-propanol (95:5) solvent system; vitamin 11 (3.3 mg,
46%) was collected at RV = 34 mL. Analytical sample of the vitamin
was obtained after reversed-phase HPLC (9.4 mm × 25 cm Zorbax
Eclipse XDB-C18 column, 4 mL/min) using methanol/water (97:3)
solvent system (RV = 38 mL).
(5E)-1α,25-Dihydroxy-2-methylenevitamin D3 (12). Treat-
ment of compound 8 in ether with a catalytic amount of iodine (2%
of the amount of 8), while keeping the solution under diffuse daylight
for 1 h, resulted in its partial isomerization. A mixture of (5Z)- and
(5E)-isomers 8 and 12 was formed in a ratio of 3:7, respectively.
Compounds were separated by reversed-phase HPLC (9.4 mm × 25
cm Zorbax Eclipse XDB-C18 column, 4 mL/min) using methanol/
water (84:16) solvent system, and analytically pure (5E)-isomer 12
was eluted at RV = 57 mL.
(5E,20S)-1α,25-Dihydroxy-2-methylenevitamin D3 (13). Anal-
ogously, as in the case of 8, iodine-catalyzed isomerization of (5Z)-
vitamin 9 to the respective (5E)-isomer 13 was performed. Analytically
H
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pure sample of the vitamin 13 was obtained after reversed-phase
HPLC (9.4 mm × 25 mm Zorbax Eclipse XDB-C18 column, 4 mL/
min) using methanol/water (84:16) solvent system (RV = 55 mL).
Biological Studies. 1. In Vitro Studies. VDR binding, HL-60
differentiation and 24-hydroxylase transcription assays were performed
as previously described.18,25
ABBREVIATIONS USED
■
1α,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; 2MD, (20S)-
1α,25-dihydroxy-2-methylene-19-norvitamin D3; VDR, vitamin
D receptor
2. In Vivo Studies. 2.1. Bone Calcium Mobilization and Intestinal
Calcium Transport. Male, weanling Sprague-Dawley rats were
purchased from Harlan (Indianapolis, IN). The animals were group
housed and placed on diet 11 (0.47% Ca) + AEK oil for 1 week
followed by diet 11 (0.02% Ca) + AEK oil for 3 weeks. The rats were
then switched to a diet containing 0.47% Ca 26 for 1 week followed by
2 weeks on a diet containing 0.02% Ca. Dose administration began
during the last week on 0.02% Ca diet. Four consecutive intra-
peritoneal doses were given approximately 24 h apart. Twenty-four
hours after the last dose, blood was collected from the severed neck
and the concentration of serum calcium determined as a measure of
bone calcium mobilization. The first 10 cm of the intestine was also
collected for the intestinal calcium transport analysis using the everted
gut sac method.25
All animals were managed in accordance with University of
Wisconsin standards and protocols for animal care and use. Our
experiments were approved by the College of Agricultural and Life
Sciences Institutional Animal Care and Use Committee.
2.2. Statistical Analysis. All statistical analyses were done using the
SAS mixed model procedures and Tukey’s pairwise comparisons (SAS
Institute, Inc. Cary, N.C., USA).
Molecular Modeling. The molecular mechanics studies were used
to establish the energy-minimized structures of the synthesized
compounds shown in Figure 2. The calculation of optimized
geometries and steric energies was initially carried out using the
algorithm from the MM+ HyperChem (release 8.0) software package
(Autodesk, Inc.). MM+ is an all-atom force field based on the MM2
functional form. The procedure used for finding the global minimum
structures involved the Conformational Search module; these
structures were further energy-minimized using the semiempirical
AM1 method. Finally, the respective vicinal proton−proton coupling
constants in the preferred, energy-minimized conformations were
calculated using PCMODEL (release 9.0) software package (Serena
Software).
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01295.
Purity criteria of the vitamin D analogues 8−13, their 1H
and 13C NMR spectra, figures with either competitive
binding curves or dose−response curves derived from
cellular differentiation and transcriptional assays, and
spectral data of the all synthesized compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
*Telephone: 608-262-1620. Fax: 608-262-7122. E-mail:
deluca@biochem.wisc.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was supported in part by funds from the Wisconsin
Alumni Research Foundation. We thank Jean Prahl and William
Blaser for their assistance on tissue culture work and
conducting VDR binding and HL-60 differentiation studies
and Erin Gudmundson and Suzanne Hanson for conducting
the in vivo studies.
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